Antibodies binding To HLA-A2/WT1

ABSTRACT

The present invention generally relates to antibodies that bind to HLA-A2/WT1, including bispecific antigen binding molecules e.g. for activating T cells. In addition, the present invention relates to polynucleotides encoding such antibodies, and vectors and host cells comprising such polynucleotides. The invention further relates to methods for producing the antibodies, and to methods of using them in the treatment of disease.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to European Patent Application No. EP17209205.8, filed Dec. 21, 2017, the disclosure of which is incorporatedherein by reference in its entirety.

SEQUENCE LISTING

The present application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Dec. 13, 2018, isnamed P34563-US ST25.txt and is 95,833 bytes in size.

FIELD OF THE INVENTION

The present invention generally relates to antibodies that bind toHLA-A2/WT1, including bispecific antigen binding molecules e.g. foractivating T cells. In addition, the present invention relates topolynucleotides encoding such antibodies, and vectors and host cellscomprising such polynucleotides. The invention further relates tomethods for producing the antibodies, and to methods of using them inthe treatment of disease.

BACKGROUND

WT1 (Wilms tumor 1, Wilms tumor protein) is an oncogenic transcriptionfactor involved in cell proliferation, differentiation, as well asapoptosis and organ development, whose expression in normal adult tissueis rare (Hinrichs and Restifo, Nat Biotechnol (2013) 31, 999-1008). WT1is, however, reported to be overexpressed in several types ofhaematological maligancies and a wide range of solid tumors (VanDriessche et al., Oncologist (2012) 17, 250-259). WT1 is a nuclearprotein, localized intracellularly. Intracellular protein can bedegraded in the proteasome, processed and presented on the cell surfaceby major histocompatibility complex (MHC) I as T cell epitopes, andrecognized by T cell receptors (TCR). As such, WT1-derived peptides suchas WT1_(RMF) (RMFPNAPYL) and WT1_(VLD) (VLDFAPPGA) are presented in thecontext of HLA-A2 on the cell surface and can trigger T cellrecognition.

Several approaches have been taken to exploit WT1 as target for cancer(immuno)therapy, including the development of cancer vaccines based onWT1-derived peptide and adoptive T cell transfer or WT1-specific Tcells. TCR-like antibodies against the HLA-A2/WT1_(RMF) complex,including bispecific derivatives thereof, have also been generated (Daoet al., Sci Transl Med (2013) 5, 176ra33; WO2012/135854; Dao et al., NatBiotechnol (2015) 33, 1079-1086; WO 2015/070061; WO 2017/060201).

Bispecific antibodies that bind to a surface antigen on target cells andan activating T cell antigen such as CD3 on T-cells (also called hereinT cell bispecific antibodies or “TCBs”) hold great promise for thetreatment of various cancers. The simultaneous binding of such anantibody to both of its targets will force a temporary interactionbetween target cell and T cell, causing crosslinking of the T cellreceptor and subsequent activation of any cytotoxic T cell andsubsequent lysis of the target cell. Given their potency in target cellkilling, the choice of target and the specificity of the targetingantibody is of utmost importance for T cell bispecific antibodies toavoid on- and off-target toxicities. Intracellular proteins such as WT1represent attractive targets, but are only accessible to T cell receptor(TCR)-like antibodies that bind major histocompatibility complex (MHC)presenting peptide antigens derived from the intracellular protein onthe cell surface. An inherent issue of TCR-like antibodies is potentialcross-reactivity with MHC molecules per se, or MHC molecules presentingpeptides other than the desired one, which could compromise organ ortissue selectivity.

SUMMARY OF THE INVENTION

The present invention provides novel antibodies, including bispecificantibodies, that bind HLA-A2/WT1 and have particularly favorableproperties for therapeutic purposes.

The present inventors have developed novel antibodies with unexpected,improved properties, that bind to HLA-A2/WT1. In particular, theantibody binds HLA-A2/WT1 with good affinity and remarkable specificity.Furthermore, the inventors have developed bispecific antigen bindingmolecules that bind to HLA-A2/WT1 and an activating T cell antigen,incorporating the novel HLA-A2/WT1 antibody and combine good efficacyand produceability with low toxicity and favorable pharmacokineticproperties.

In a first aspect the present invention provides an antibody that bindsto HLA-A2/WT1, wherein the antibody comprises

(i) a heavy chain variable region (VH) comprising a heavy chaincomplementary determining region (HCDR) 1 of SEQ ID NO: 1, a HCDR 2 ofSEQ ID NO: 2, and a HCDR 3 of SEQ ID NO: 3, and a light chain variableregion (VL) comprising a light chain complementarity determining region(LCDR) 1 of SEQ ID NO: 4, a LCDR 2 of SEQ ID NO: 5 and a LCDR 3 of SEQID NO: 6;(ii) a VH comprising a HCDR 1 of SEQ ID NO: 9, a HCDR 2 of SEQ ID NO:10, and a HCDR 3 of SEQ ID NO: 11, and a VL comprising a LCDR 1 of SEQID NO: 12, a LCDR 2 of SEQ ID NO: 13 and a LCDR 3 of SEQ ID NO: 14;(iii) a VH comprising a HCDR 1 of SEQ ID NO: 17, a HCDR 2 of SEQ ID NO:18, and a HCDR 3 of SEQ ID NO: 19, and a VL comprising a LCDR 1 of SEQID NO: 20, a LCDR 2 of SEQ ID NO: 21 and a LCDR 3 of SEQ ID NO: 22;(iv) a VH comprising a HCDR 1 of SEQ ID NO: 25, a HCDR 2 of SEQ ID NO:26, and a HCDR 3 of SEQ ID NO: 27, and a VL comprising a LCDR 1 of SEQID NO: 28, a LCDR 2 of SEQ ID NO: 29 and a LCDR 3 of SEQ ID NO: 30;(v) a VH comprising a HCDR 1 of SEQ ID NO: 33, a HCDR 2 of SEQ ID NO:34, and a HCDR 3 of SEQ ID NO: 35, and a VL comprising a LCDR 1 of SEQID NO: 36, a LCDR 2 of SEQ ID NO: 37 and a LCDR 3 of SEQ ID NO: 38;(vi) a VH comprising a HCDR 1 of SEQ ID NO: 41, a HCDR 2 of SEQ ID NO:42, and a HCDR 3 of SEQ ID NO: 43, and a VL comprising a LCDR 1 of SEQID NO: 44, a LCDR 2 of SEQ ID NO: 45 and a LCDR 3 of SEQ ID NO: 46;(vii) a VH comprising a HCDR 1 of SEQ ID NO: 49, a HCDR 2 of SEQ ID NO:50, and a HCDR 3 of SEQ ID NO: 51, and a VL comprising a LCDR 1 of SEQID NO: 52, a LCDR 2 of SEQ ID NO: 53 and a LCDR 3 of SEQ ID NO: 54;(viii) a VH comprising a HCDR 1 of SEQ ID NO: 57, a HCDR 2 of SEQ ID NO:58, and a HCDR 3 of SEQ ID NO: 59, and a VL comprising a LCDR 1 of SEQID NO: 60, a LCDR 2 of SEQ ID NO: 61 and a LCDR 3 of SEQ ID NO: 62; or(ix) a VH comprising a HCDR 1 of SEQ ID NO: 65, a HCDR 2 of SEQ ID NO:66, and a HCDR 3 of SEQ ID NO: 67, and a VL comprising a LCDR 1 of SEQID NO: 68, a LCDR 2 of SEQ ID NO: 69 and a LCDR 3 of SEQ ID NO: 70.

In one embodiment, the antibody comprises

(i) a VH comprising an amino acid sequence that is at least about 95%,96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQID NO: 7, and a VL comprising an amino acid sequence that is at leastabout 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acidsequence of SEQ ID NO: 8;(ii) a VH comprising an amino acid sequence that is at least about 95%,96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQID NO: 15, and a VL comprising an amino acid sequence that is at leastabout 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acidsequence of SEQ ID NO: 16;(iii) a VH comprising an amino acid sequence that is at least about 95%,96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQID NO: 23, and a VL comprising an amino acid sequence that is at leastabout 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acidsequence of SEQ ID NO: 24;(iv) a VH comprising an amino acid sequence that is at least about 95%,96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQID NO: 31, and a VL comprising an amino acid sequence that is at leastabout 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acidsequence of SEQ ID NO: 32;(v) a VH comprising an amino acid sequence that is at least about 95%,96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQID NO: 39, and a VL comprising an amino acid sequence that is at leastabout 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acidsequence of SEQ ID NO: 40;(vi) a VH comprising an amino acid sequence that is at least about 95%,96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQID NO: 47, and a VL comprising an amino acid sequence that is at leastabout 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acidsequence of SEQ ID NO: 48;(vii) a VH comprising an amino acid sequence that is at least about 95%,96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQID NO: 55, and a VL comprising an amino acid sequence that is at leastabout 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acidsequence of SEQ ID NO: 56;(viii) a VH comprising an amino acid sequence that is at least about95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence ofSEQ ID NO: 63, and a VL comprising an amino acid sequence that is atleast about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acidsequence of SEQ ID NO: 64; or(ix) a VH comprising an amino acid sequence that is at least about 95%,96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQID NO: 71, and a VL comprising an amino acid sequence that is at leastabout 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acidsequence of SEQ ID NO: 72.

In one embodiment, the antibody is an IgG, particularly an IgG₁,antibody. In one embodiment, the antibody is a full-length antibody. Inanother embodiment, the antibody is an antibody fragment selected fromthe group of an Fv molecule, a scFv molecule, a Fab molecule, and aF(ab′)₂ molecule.

In one embodiment, the antibody is a multispecific antibody.

The invention also provides a bispecific antigen binding molecule,comprising

(a) a first antigen binding moiety that binds to a first antigen,wherein the first antigen is HLA-A2/WT1 and the first antigen bindingmoiety comprises(i) a heavy chain variable region (VH) comprising a heavy chaincomplementary determining region (HCDR) 1 of SEQ ID NO: 1, a HCDR 2 ofSEQ ID NO: 2, and a HCDR 3 of SEQ ID NO: 3, and a light chain variableregion (VL) comprising a light chain complementarity determining region(LCDR) 1 of SEQ ID NO: 4, a LCDR 2 of SEQ ID NO: 5 and a LCDR 3 of SEQID NO: 6;(ii) a VH comprising a HCDR 1 of SEQ ID NO: 9, a HCDR 2 of SEQ ID NO:10, and a HCDR 3 of SEQ ID NO: 11, and a VL comprising a LCDR 1 of SEQID NO: 12, a LCDR 2 of SEQ ID NO: 13 and a LCDR 3 of SEQ ID NO: 14;(iii) a VH comprising a HCDR 1 of SEQ ID NO: 17, a HCDR 2 of SEQ ID NO:18, and a HCDR 3 of SEQ ID NO: 19, and a VL comprising a LCDR 1 of SEQID NO: 20, a LCDR 2 of SEQ ID NO: 21 and a LCDR 3 of SEQ ID NO: 22;(iv) a VH comprising a HCDR 1 of SEQ ID NO: 25, a HCDR 2 of SEQ ID NO:26, and a HCDR 3 of SEQ ID NO: 27, and a VL comprising a LCDR 1 of SEQID NO: 28, a LCDR 2 of SEQ ID NO: 29 and a LCDR 3 of SEQ ID NO: 30;(v) a VH comprising a HCDR 1 of SEQ ID NO: 33, a HCDR 2 of SEQ ID NO:34, and a HCDR 3 of SEQ ID NO: 35, and a VL comprising a LCDR 1 of SEQID NO: 36, a LCDR 2 of SEQ ID NO: 37 and a LCDR 3 of SEQ ID NO: 38;(vi) a VH comprising a HCDR 1 of SEQ ID NO: 41, a HCDR 2 of SEQ ID NO:42, and a HCDR 3 of SEQ ID NO: 43, and a VL comprising a LCDR 1 of SEQID NO: 44, a LCDR 2 of SEQ ID NO: 45 and a LCDR 3 of SEQ ID NO: 46;(vii) a VH comprising a HCDR 1 of SEQ ID NO: 49, a HCDR 2 of SEQ ID NO:50, and a HCDR 3 of SEQ ID NO: 51, and a VL comprising a LCDR 1 of SEQID NO: 52, a LCDR 2 of SEQ ID NO: 53 and a LCDR 3 of SEQ ID NO: 54;(viii) a VH comprising a HCDR 1 of SEQ ID NO: 57, a HCDR 2 of SEQ ID NO:58, and a HCDR 3 of SEQ ID NO: 59, and a VL comprising a LCDR 1 of SEQID NO: 60, a LCDR 2 of SEQ ID NO: 61 and a LCDR 3 of SEQ ID NO: 62; or(ix) a VH comprising a HCDR 1 of SEQ ID NO: 65, a HCDR 2 of SEQ ID NO:66, and a HCDR 3 of SEQ ID NO: 67, and a VL comprising a LCDR 1 of SEQID NO: 68, a LCDR 2 of SEQ ID NO: 69 and a LCDR 3 of SEQ ID NO: 70; and(b) a second antigen binding moiety which specifically binds to a secondantigen.

In one embodiment, the first antigen binding moiety comprises

(i) a VH comprising an amino acid sequence that is at least about 95%,96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQID NO: 7, and a VL comprising an amino acid sequence that is at leastabout 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acidsequence of SEQ ID NO: 8;(ii) a VH comprising an amino acid sequence that is at least about 95%,96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQID NO: 15, and a VL comprising an amino acid sequence that is at leastabout 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acidsequence of SEQ ID NO: 16;(iii) a VH comprising an amino acid sequence that is at least about 95%,96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQID NO: 23, and a VL comprising an amino acid sequence that is at leastabout 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acidsequence of SEQ ID NO: 24;(iv) a VH comprising an amino acid sequence that is at least about 95%,96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQID NO: 31, and a VL comprising an amino acid sequence that is at leastabout 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acidsequence of SEQ ID NO: 32;(v) a VH comprising an amino acid sequence that is at least about 95%,96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQID NO: 39, and a VL comprising an amino acid sequence that is at leastabout 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acidsequence of SEQ ID NO: 40;(vi) a VH comprising an amino acid sequence that is at least about 95%,96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQID NO: 47, and a VL comprising an amino acid sequence that is at leastabout 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acidsequence of SEQ ID NO: 48;(vii) a VH comprising an amino acid sequence that is at least about 95%,96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQID NO: 55, and a VL comprising an amino acid sequence that is at leastabout 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acidsequence of SEQ ID NO: 56;(viii) a VH comprising an amino acid sequence that is at least about95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence ofSEQ ID NO: 63, and a VL comprising an amino acid sequence that is atleast about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acidsequence of SEQ ID NO: 64; or(ix) a VH comprising an amino acid sequence that is at least about 95%,96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQID NO: 71, and a VL comprising an amino acid sequence that is at leastabout 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acidsequence of SEQ ID NO: 72.

In one embodiment, the second antigen is CD3, particularly CD3ε. In oneembodiment, the second antigen binding moiety comprises a VH comprisinga HCDR 1 of SEQ ID NO: 115, a HCDR 2 of SEQ ID NO: 116, and a HCDR 3 ofSEQ ID NO: 117, and a VL comprising a LCDR 1 of SEQ ID NO: 118, a LCDR 2of SEQ ID NO: 119 and a LCDR 3 of SEQ ID NO: 120. In one embodiment, theVH of the second antigen binding moiety comprises an amino acid sequencethat is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to theamino acid sequence of SEQ ID NO: 121, and the VL of the second antigenbinding moiety comprises an amino acid sequence that is at least about95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence ofSEQ ID NO: 122. In one embodiment, the first and/or the second antigenbinding moiety is a Fab molecule. In one embodiment, the second antigenbinding moiety is a Fab molecule wherein the variable domains VL and VHor the constant domains CL and CH1, particularly the variable domains VLand VH, of the Fab light chain and the Fab heavy chain are replaced byeach other.

In one embodiment, the first antigen binding moiety is a Fab moleculewherein in the constant domain the amino acid at position 124 issubstituted independently by lysine (K), arginine (R) or histidine (H)(numbering according to Kabat) and the amino acid at position 123 issubstituted independently by lysine (K), arginine (R) or histidine (H)(numbering according to Kabat), and in the constant domain CH1 the aminoacid at position 147 is substituted independently by glutamic acid (E),or aspartic acid (D) (numbering according to Kabat EU index) and theamino acid at position 213 is substituted independently by glutamic acid(E), or aspartic acid (D) (numbering according to Kabat EU index). Inone embodiment, the first and the second antigen binding moiety arefused to each other, optionally via a peptide linker. In one embodiment,the first and the second antigen binding moiety are each a Fab moleculeand either (i) the second antigen binding moiety is fused at theC-terminus of the Fab heavy chain to the N-terminus of the Fab heavychain of the first antigen binding moiety, or (ii) the first antigenbinding moiety is fused at the C-terminus of the Fab heavy chain to theN-terminus of the Fab heavy chain of the second antigen binding moiety.

In one embodiment, the bispecific antigen binding molecule comprises athird antigen binding moiety. In one embodiment, the third antigenmoiety is identical to the first antigen binding moiety.

In one embodiment, the bispecific antigen binding molecule comprises anFc domain composed of a first and a second subunit. In one embodiment,the first, the second and, where present, the third antigen bindingmoiety are each a Fab molecule; and either (i) the second antigenbinding moiety is fused at the C-terminus of the Fab heavy chain to theN-terminus of the Fab heavy chain of the first antigen binding moietyand the first antigen binding moiety is fused at the C-terminus of theFab heavy chain to the N-terminus of the first subunit of the Fc domain,or (ii) the first antigen binding moiety is fused at the C-terminus ofthe Fab heavy chain to the N-terminus of the Fab heavy chain of thesecond antigen binding moiety and the second antigen binding moiety isfused at the C-terminus of the Fab heavy chain to the N-terminus of thefirst subunit of the Fc domain; and the third antigen binding moiety,where present, is fused at the C-terminus of the Fab heavy chain to theN-terminus of the second subunit of the Fc domain. In one embodiment,the Fc domain is an IgG, particularly an IgG₁, Fc domain. In oneembodiment, the Fc domain is a human Fc domain. In one embodiment, anamino acid residue in the CH3 domain of the first subunit of the Fcdomain is replaced with an amino acid residue having a larger side chainvolume, thereby generating a protuberance within the CH3 domain of thefirst subunit which is positionable in a cavity within the CH3 domain ofthe second subunit, and an amino acid residue in the CH3 domain of thesecond subunit of the Fc domain is replaced with an amino acid residuehaving a smaller side chain volume, thereby generating a cavity withinthe CH3 domain of the second subunit within which the protuberancewithin the CH3 domain of the first subunit is positionable. In oneembodiment, the Fc domain comprises one or more amino acid substitutionthat reduces binding to an Fc receptor and/or effector function.

According to another aspect of the invention there is provided one ormore isolated polynucleotide(s) encoding an antibody or bispecificantigen binding molecule of the invention. The invention furtherprovides one or more expression vector(s) comprising the isolatedpolynucleotide(s) of the invention, and a host cell comprising theisolated polynucleotide(s) or the expression vector(s) of the invention.In some embodiments the host cell is a eukaryotic cell, particularly amammalian cell.

In another aspect is provided a method of producing an antibody thatbinds to HLA-A2/WT1, comprising the steps of a) culturing the host cellof the invention under conditions suitable for the expression of theantibody and b) recovering the antibody. The invention also encompassesan antibody that binds to HLA-A2/WT1 produced by the method of theinvention.

The invention further provides a pharmaceutical composition comprisingthe antibody or bispecific antigen binding molecule of the invention anda pharmaceutically acceptable carrier.

Also encompassed by the invention are methods of using the antibody,bispecific antigen binding molecule and pharmaceutical composition ofthe invention. In one aspect the invention provides an antibody,bispecific antigen binding molecule or pharmaceutical compositionaccording to the invention for use as a medicament. In one aspect isprovided an antibody, bispecific antigen binding molecule orpharmaceutical composition according to the invention for use in thetreatment of a disease. In a specific embodiment the disease is cancer.

Also provided is the use of an antibody or bispecific antigen bindingmolecule according to the invention in the manufacture of a medicamentfor the treatment of a disease; as well as a method of treating adisease in an individual, comprising administering to said individual atherapeutically effective amount of a composition comprising theantibody or bispecific antigen binding molecule according to theinvention in a pharmaceutically acceptable form. In a specificembodiment the disease is cancer. In any of the above embodiments theindividual preferably is a mammal, particularly a human.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-Z. Exemplary configurations of the bispecific antigen bindingmolecules of the invention. (A, D) Illustration of the “1+1 CrossMab”molecule. (B, E) Illustration of the “2+1 IgG Crossfab” molecule withalternative order of Crossfab and Fab components (“inverted”). (C, F)Illustration of the “2+1 IgG Crossfab” molecule. (G, K) Illustration ofthe “1+1 IgG Crossfab” molecule with alternative order of Crossfab andFab components (“inverted”). (H, L) Illustration of the “1+1 IgGCrossfab” molecule. (I, M) Illustration of the “2+1 IgG Crossfab”molecule with two CrossFabs. (J, N) Illustration of the “2+1 IgGCrossfab” molecule with two CrossFabs and alternative order of Crossfaband Fab components (“inverted”). (0, S) Illustration of the“Fab-Crossfab” molecule. (P, T) Illustration of the “Crossfab-Fab”molecule. (Q, U) Illustration of the “(Fab)₂-Crossfab” molecule. (R, V)Illustration of the “Crossfab-(Fab)₂” molecule. (W, Y) Illustration ofthe “Fab-(Crossfab)₂” molecule. (X, Z) Illustration of the“(Crossfab)₂-Fab” molecule. Black dot: optional modification in the Fcdomain promoting heterodimerization. ++, −−: amino acids of oppositecharges optionally introduced in the CH1 and CL domains. Crossfabmolecules are depicted as comprising an exchange of VH and VL regions,but may—in embodiments wherein no charge modifications are introduced inCH1 and CL domains—alternatively comprise an exchange of the CH1 and CLdomains.

FIG. 2. Illustration of the T-cell bispecific (TCB) antibody moleculesprepared in the Examples. All tested TCB antibody molecules wereproduced as “2+1 IgG CrossFab, inverted” with charge modifications(VH/VL exchange in CD3 binder, charge modifications in WT1 binders,EE=147E, 213E; RK=123R, 124K).

FIGS. 3A-G. Binding of HLA-A2/WT1 IgG antibodies to peptide-pulsed T2cells. (A) 11D06 IgG, (B) 33H09-IgG, (C) 11B09-IgG, (D) 13B04-IgG, (E)5E11-IgG, (F) 5C01-IgG, (G) 11G06-IgG.

FIGS. 4A-H. Activation of T cells by HLA-A2/WT1×CD3 bispecificantibodies (TCBs) upon binding to peptide-pulsed T2 cells (NFAT reporterassay). (A) 11D06-TCB, (B) 33H09-TCB, (C) 11B09-TCB, (D) 13B04-TCB, (E)ESK1-TCB, (F) 5E11-TCB. (G) 5C01-TCB, (H) DP47GS-TCB.

FIGS. 5A-F. Killing of peptide-pulsed T2 cells mediated byHLA-A2/WT1×CD3 bispecific antibodies (TCBs). (A) 11D06-TCB, (B)33H09-TCB, (C) 13B04-TCB, (D) 11B09-TCB, (E) 33F05-TCB, (F) 5C01-TCB.

FIGS. 6A-G. Killing of HLA-A2+WT1+ tumor cell lines mediated byHLA-A2/WT1×CD3 bispecific antibodies (TCBs). (A) Overview of cell lines.(B-E) Killing of cell lines by (B) 11D06-TCB, (C) 33H09-TCB, (D)11B09-TCB, (E) 13B04-TCB. (F) Killing of SKM-1 cells by different TCBs.(G) Killing of BJAB cells by different TCBs.

FIGS. 7A-B. Activation of T cells by HLA-A2/WT1×CD3 bispecificantibodies (TCBs) upon binding to HLA-A2+WT1+ tumor cell lines. (A)SKM-1 cells, (B) BJAB cells.

FIGS. 8A-F. No binding to off-target peptides by selected HLA-A2/WT1×CD3bispecific antibodies (TCBs). (A-C) Binding to peptide-pulsed T2 cellsby (A) 11D06-TCB, (B) 33H09-TCB, (C) ESK1-TCB. (D-E) Activation of Tcells upon binding to peptide-pulsed T2 cells by (D) 11D06-TCB and (E)33H09-TCB. (F) Overview of peptides.

FIGS. 9A-G. No binding to additional off-target peptides by selectedHLA-A2/WT1×CD3 bispecific antibodies (TCBs). (A-B) Activation of T cellsupon binding to peptide-pulsed T2 cells by (A) 11D06-TCB and (B)33H09-TCB. The 6 indicated off-target peptides were tested along withthe RMF peptide. (C-G) Killing of peptide-pulsed T2 cells by (C, E)11D06-TCB, (D, F) 33H09-TCB and (G) ESK1-TCB. The 19 indicatedoff-target peptides were tested along with the RMF and VLD peptides.

FIGS. 10A-B. No killing of normal bone marrow-derived CD34+ stem cellsmediated by selected HLA-A2/WT1×CD3 bispecific antibodies (TCBs). (A)11D06-TCB, (B) 33H09-TCB.

FIGS. 11A-N. Identification by alanine scan of binding residues in RMFpeptide for selected HLA-A2/WT1×CD3 bispecific antibodies (TCBs). (A)RMF native peptide, (B) RMF R1Y peptide, (C) RMF R1A peptide, (D) RMFM2A peptide, (E) RMF F3A peptide, (F) RMF P4A peptide, (G) RMF NSApeptide, (H) RMF A6G peptide, (I) RMF P7A peptide, (J) RMF Y8A peptide,(K) RMF L9A peptide. (L) Overview of peptides. (M) Fold change of EC₅₀relative to EC₅₀ for the RMF native peptide. (N) Critical contactresidues.

FIG. 12. Pharmacokinetic profile of HLA-A2/WT1×CD3 bispecific antibodies(11D06-TCB and 33H09-TCB) after single injection in NSG mice.

FIGS. 13A-G. Efficacy study with HLA-A2/WT1×CD3 bispecific antibodies(“TCBs”) in SKM-1 xenograft in humanized mice. (A) Study design. (B)Treatment groups. (C) Tumor growth kinetics (mean) in all treatmentgroups. (D) Single tumor growth kinetics in the vehicle group. (E)Single tumor growth kinetics in the 11D06-TCB group. (F) Single tumorgrowth kinetics in the 33H09-TCB group. (G) Statistics. Calculationsbased on day 38 (vehicle as control group). Tumor growth inhibition(TGI): TGI>100→tumor regression, TGI=100→tumor stasis. Treatment tocontrol ratio (TCR): TCR=1→no effect, TCR=0→complete regression.

FIGS. 14A-C. Overview of crystal structures of HLA-A2/WT1 antibody—pMHCcomplexes. The antibodies (Fab fragments) are shown on top, with theheavy chain colored dark gray and the light chain colored light gray.Crystallized solvent atoms are not shown. (A) 1.98 Å resolution crystalstructure of 5C01 Fab in complex with HLA-A02/VLD pMHC. Fab-pMHC contactarea: 476≈Å², peptide contribution: 68≈Å². (B) 2.60 Å resolution crystalstructure of 11D06 Fab in complex with HLA-A02/RMF pMHC. Fab-pMHCcontact area: 397≈Å², peptide contribution: 107≈Å². (C) 3.05 Åresolution crystal structure of ESK1 Fab in complex with HLA-A02/RMFpMHC (published, PDB ID 4WUU). Fab-pMHC contact area: 505≈Å², peptidecontribution: 60≈Å².

FIG. 15. Close-up view on the 5C01 Fab-HLA-A2/WT1_(V)LD pMHC bindinginterface. Essential chemical interactions between Fab and pMHC asidentified by BIOVIA Discovery Studio 4.5 are highlighted. Solvent atomsare not shown.

FIG. 16. Interface and interaction matrix of 5C01 Fab residues (rows)with HLA-A2/WT1_(VLD) pMHC residues (columns). N=near/neighboring,H═H-bond, Pi=Pi interactions, SB=salt bridge. Interface residues definedas residues that undergo a change in solvent-accessible surface area inabsence/presence of the interaction partner.

FIG. 17. Close-up view on the 11D06 Fab-HLA-A2/WT1_(RMF) pMHC bindinginterface. Essential chemical interactions between Fab and pMHC asidentified by BIOVIA Discovery Studio 4.5 are highlighted. Solvent atomsare not shown.

FIG. 18. Interface and interaction matrix of 11D06 Fab residues (rows)with HLA-A2/WT1_(RMF) pMHC residues (columns). N=near/neighboring,H═H-bond, Pi=Pi interactions, SB=salt bridge. Interface residues definedas residues that undergo a change in solvent-accessible surface area inabsence/presence of the interaction partner.

FIG. 19. Close-up view on the ESK1 Fab-HLA-A2/WT1_(RMF) pMHC bindinginterface (PDB ID 4WUU). Essential chemical interactions between Fab andpMHC as identified by BIOVIA Discovery Studio 4.5 are highlighted.Solvent atoms are not shown.

FIG. 20. Interface and interaction matrix of ESK1 Fab residues (rows)with HLA-A2/WT1_(RMF) pMHC residues (columns). N=near/neighboring,H═H-bond, Pi=Pi interactions, SB=salt bridge. Interface residues definedas residues that undergo a change in solvent-accessible surface area inabsence/presence of the interaction partner.

FIG. 21. Killing of HLA-A2+/WT1+SKM-1 cells mediated by HLA-A2/WT1×CD3bispecific antibodies with different CD3 binders.

FIG. 22. Killing of RMF peptide-pulsed T2 cells mediated byHLA-A2/WT1×CD3 bispecific antibodies (TCBs).

FIGS. 23A-D. Assessment of binding to off-target peptides byHLA-A2/WT1×CD3 bispecific antibodies (TCBs) Aali-TCB (A), Daniel-TCB(B), ESK1-TCB (C) and 11D06-TCB (D).

FIG. 24. Pharmacokinetic profile of HLA-A2/WT1×CD3 bispecific antibody11D06-TCB (V9) after single injection in NSG mice.

FIGS. 25A-D. Efficacy study with HLA-A2/WT1×CD3 bispecific antibody11D06-TCB (V9) in SKM-1 xenograft in humanized mice. (A) Tumor growthkinetics (mean) in all treatment groups. (B) Single tumor growthkinetics in the vehicle group. (C) Single tumor growth kinetics in the11D06-TCB (V9) group. (D) Statistics. Calculations based on day 48(vehicle as control group). Tumor growth inhibition (TGI): TGI>100→tumorregression, TGI=100→tumor stasis. Treatment to control ratio (TCR):TCR=1→no effect, TCR=0→complete regression.

FIG. 26. Activation of T cells by HLA-A2/WT1×CD3 bispecific antibodies(TCBs) upon binding to CHO-K1 cells expressing a HLA-A02/WT1_(RMF) pMHCcomplex (NFAT reporter assay). Solid line: 11D06-TCB (V9) (“2+1”format). Dashed line: analogous molecule (11D06 and V9 binders) in “1+1CrossMab” format.

DETAILED DESCRIPTION OF THE INVENTION Definitions

Terms are used herein as generally used in the art, unless otherwisedefined in the following.

As used herein, the term “antigen binding molecule” refers in itsbroadest sense to a molecule that specifically binds an antigenicdeterminant. Examples of antigen binding molecules are immunoglobulinsand derivatives, e.g. fragments, thereof.

The term “bispecific” means that the antigen binding molecule is able tospecifically bind to at least two distinct antigenic determinants.Typically, a bispecific antigen binding molecule comprises two antigenbinding sites, each of which is specific for a different antigenicdeterminant.

In certain embodiments the bispecific antigen binding molecule iscapable of simultaneously binding two antigenic determinants,particularly two antigenic determinants expressed on two distinct cells.

The term “valent” as used herein denotes the presence of a specifiednumber of antigen binding sites in an antigen binding molecule. As such,the term “monovalent binding to an antigen” denotes the presence of one(and not more than one) antigen binding site specific for the antigen inthe antigen binding molecule.

An “antigen binding site” refers to the site, i.e. one or more aminoacid residues, of an antigen binding molecule which provides interactionwith the antigen. For example, the antigen binding site of an antibodycomprises amino acid residues from the complementarity determiningregions (CDRs). A native immunoglobulin molecule typically has twoantigen binding sites, a Fab molecule typically has a single antigenbinding site.

As used herein, the term “antigen binding moiety” refers to apolypeptide molecule that specifically binds to an antigenicdeterminant. In one embodiment, an antigen binding moiety is able todirect the entity to which it is attached (e.g. a second antigen bindingmoiety) to a target site, for example to a specific type of tumor cellbearing the antigenic determinant. In another embodiment an antigenbinding moiety is able to activate signaling through its target antigen,for example a T cell receptor complex antigen. Antigen binding moietiesinclude antibodies and fragments thereof as further defined herein.Particular antigen binding moieties include an antigen binding domain ofan antibody, comprising an antibody heavy chain variable region and anantibody light chain variable region. In certain embodiments, theantigen binding moieties may comprise antibody constant regions asfurther defined herein and known in the art. Useful heavy chain constantregions include any of the five isotypes: α, δ, ε, γ, or μ. Useful lightchain constant regions include any of the two isotypes: κ and λ.

As used herein, the term “antigenic determinant” or “antigen” refers toa site on a polypeptide macromolecule to which an antigen binding moietybinds, forming an antigen binding moiety-antigen complex. Usefulantigenic determinants can be found, for example, on the surfaces oftumor cells, on the surfaces of virus-infected cells, on the surfaces ofother diseased cells, on the surface of immune cells, free in bloodserum, and/or in the extracellular matrix (ECM).

The term “epitope” denotes the site on an antigen, either proteinaceousor non-proteinaceous, to which an antigen binding moiety binds. Epitopescan be formed both from contiguous amino acid stretches (linear epitope)or comprise non-contiguous amino acids (conformational epitope), e.g.coming in spatial proximity due to the folding of the antigen, i.e. bythe tertiary folding of a proteinaceous antigen. Linear epitopes aretypically still bound by an antigen binding moiety after exposure of theproteinaceous antigen to denaturing agents, whereas conformationalepitopes are typically destroyed upon treatment with denaturing agents.An epitope comprises at least 3, at least 4, at least 5, at least 6, atleast 7, or 8-10 amino acids in a unique spatial conformation.

“CD3” refers to any native CD3 from any vertebrate source, includingmammals such as primates (e.g. humans), non-human primates (e.g.cynomolgus monkeys) and rodents (e.g. mice and rats), unless otherwiseindicated. The term encompasses “full-length,” unprocessed CD3 as wellas any form of CD3 that results from processing in the cell. The termalso encompasses naturally occurring variants of CD3, e.g., splicevariants or allelic variants. In one embodiment, CD3 is human CD3,particularly the epsilon subunit of human CD3 (CD3ε). The amino acidsequence of human CD3ε is shown in UniProt (www.uniprot.org) accessionno. P07766 (version 189), or NCBI (www.ncbi.nlm.nih.gov/) RefSeqNP_000724.1. See also SEQ ID NO: 107. The amino acid sequence ofcynomolgus [Macaca fascicularis] CD3ε is shown in NCBI GenBank no.BAB71849.1. See also SEQ ID NO: 108.

“WT1”, also known as “Wilms tumor 1” or “Wilms tumor protein”, refers toany native WT1 from any vertebrate source, including mammals such asprimates (e.g. humans), non-human primates (e.g. cynomolgus monkeys) androdents (e.g. mice and rats), unless otherwise indicated. The termencompasses “full-length,” unprocessed WT1 as well as any form of WT1that results from processing in the cell. The term also encompassesnaturally occurring variants of WT1, e.g., splice variants or allelicvariants. In one embodiment, WT1 is human WT1, particularly the proteinof SEQ ID NO: 106. Human WT1 is described in UniProt (www.uniprot.org)accession no. P19544 (entry version 215), and an amino acid sequence ofhuman WT1 is also shown in SEQ ID NO: 106.

By “VLD”, “VLD peptide” or “WT1_(VLD)” is meant the WT1 derived peptidehaving the amino acid sequence VLDFAPPGA (SEQ ID NO: 77; position 37-45of the WT1 protein of SEQ ID NO: 106).

By “RMF”, “RMF peptide” or “WT1_(RMF)” is meant the WT1 derived peptidehaving the amino acid sequence RMFRNAPYL (SEQ ID NO: 78; position126-134 of the WT1 protein of SEQ ID NO: 106).

“HLA-A2”, “HLA-A*02”, “HLA-A02”, or “HLA-A*2” (used interchangeably)refers to a human leukocyte antigen serotype in the HLA-A serotypegroup. The HLA-A2 protein (encoded by the respective HLA gene)constitutes the a chain of the respective class I MHC (majorhistocompatibility complex) protein, which further comprises a (32microglobulin subunit. A specific HLA-A2 protein is HLA-A201 (alsoreferred to as HLA-A0201, HLA-A02.01, or HLA-A*02:01). In specificembodiments, the HLA-A2 protein described herein is HLA-A201“HLA-A2/WT1” refers to a complex of a HLA-A2 molecule and a WT1 derivedpeptide (also referred to herein as a “WT1 peptide”), specifically theRMF or VLD peptide (“HLA-A2/WT1_(RMF)” and “HLA-A2/WT1_(VLD)”,respectively). The antibody or bispecific antigen binding molecule ofthe present invention specifically binds to either the HLA-A2/WT1_(RMF)or the HLA-A2/WT1_(VLD) complex.

By “specific binding” is meant that the binding is selective for theantigen and can be discriminated from unwanted or non-specificinteractions. The ability of an antigen binding moiety to bind to aspecific antigenic determinant can be measured either through anenzyme-linked immunosorbent assay (ELISA) or other techniques familiarto one of skill in the art, e.g. surface plasmon resonance (SPR)technique (analyzed e.g. on a BIAcore instrument) (Liljeblad et al.,Glyco J 17, 323-329 (2000)), and traditional binding assays (Heeley,Endocr Res 28, 217-229 (2002)). Suitable assays for determining thespecificity of the antibody and bispecific antigen binding molecule ofthe present invention are described herein, e.g. in Examples 4, 9 and 10hereinbelow. In one embodiment, the extent of binding of an antigenbinding moiety to an unrelated protein is less than about 10% of thebinding of the antigen binding moiety to the antigen as measured, e.g.,by SPR. In certain embodiments, an antigen binding moiety that binds tothe antigen, or an antigen binding molecule comprising that antigenbinding moiety, has a dissociation constant (K_(D)) of ≤1 μM, ≤100 nM,≤10 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM, or ≤0.001 nM (e.g. 10⁻⁸ M or less,e.g. from 10⁻⁸M to 10⁻¹³M, e.g., from 10⁻⁹ M to 10⁻¹³ M).

“Affinity” refers to the strength of the sum total of non-covalentinteractions between a single binding site of a molecule (e.g., areceptor) and its binding partner (e.g., a ligand). Unless indicatedotherwise, as used herein, “binding affinity” refers to intrinsicbinding affinity which reflects a 1:1 interaction between members of abinding pair (e.g., an antigen binding moiety and an antigen, or areceptor and its ligand). The affinity of a molecule X for its partner Ycan generally be represented by the dissociation constant (K_(D)), whichis the ratio of dissociation and association rate constants (k_(off) andk_(on), respectively). Thus, equivalent affinities may comprisedifferent rate constants, as long as the ratio of the rate constantsremains the same. Affinity can be measured by well established methodsknown in the art, including those described herein. A particular methodfor measuring affinity is Surface Plasmon Resonance (SPR).

“Reduced binding”, for example reduced binding to an Fc receptor, refersto a decrease in affinity for the respective interaction, as measuredfor example by SPR. For clarity, the term includes also reduction of theaffinity to zero (or below the detection limit of the analytic method),i.e. complete abolishment of the interaction. Conversely, “increasedbinding” refers to an increase in binding affinity for the respectiveinteraction.

An “activating T cell antigen” as used herein refers to an antigenicdeterminant expressed on the surface of a T lymphocyte, particularly acytotoxic T lymphocyte, which is capable of inducing T cell activationupon interaction with an antigen binding molecule. Specifically,interaction of an antigen binding molecule with an activating T cellantigen may induce T cell activation by triggering the signaling cascadeof the T cell receptor complex. In a particular embodiment theactivating T cell antigen is CD3, particularly the epsilon subunit ofCD3 (see UniProt no. P07766 (version 189), NCBI RefSeq no. NP_000724.1,SEQ ID NO: 107 for the human sequence; or UniProt no. Q95LI5 (version49), NCBI GenBank no. BAB71849.1, SEQ ID NO: 108 for the cynomolgus[Macaca fascicularis] sequence).

“T cell activation” as used herein refers to one or more cellularresponse of a T lymphocyte, particularly a cytotoxic T lymphocyte,selected from: proliferation, differentiation, cytokine secretion,cytotoxic effector molecule release, cytotoxic activity, and expressionof activation markers. Suitable assays to measure T cell activation areknown in the art and described herein.

A “target cell antigen” as used herein refers to an antigenicdeterminant presented on the surface of a target cell, for example acell in a tumor such as a cancer cell or a cell of the tumor stroma. Ina particular embodiment, the target cell antigen is HLA-A2/WT1,particularly HLA-A2/WT1_(RMF) or HLA-A2/WT1_(VLD), most particularlyHLA-A2/WT1_(RMF).

As used herein, the terms “first”, “second” or “third” with respect toFab molecules etc., are used for convenience of distinguishing whenthere is more than one of each type of moiety. Use of these terms is notintended to confer a specific order or orientation of the bispecificantigen binding molecule unless explicitly so stated.

By “fused” is meant that the components (e.g. a Fab molecule and an Fcdomain subunit) are linked by peptide bonds, either directly or via oneor more peptide linkers.

A “Fab molecule” refers to a protein consisting of the VH and CH1 domainof the heavy chain (the “Fab heavy chain”) and the VL and CL domain ofthe light chain (the “Fab light chain”) of an immunoglobulin.

By a “crossover” Fab molecule (also termed “Crossfab”) is meant a Fabmolecule wherein the variable domains or the constant domains of the Fabheavy and light chain are exchanged (i.e. replaced by each other), i.e.the crossover Fab molecule comprises a peptide chain composed of thelight chain variable domain VL and the heavy chain constant domain 1 CH1(VL-CH1, in N- to C-terminal direction), and a peptide chain composed ofthe heavy chain variable domain VH and the light chain constant domainCL (VH-CL, in N- to C-terminal direction). For clarity, in a crossoverFab molecule wherein the variable domains of the Fab light chain and theFab heavy chain are exchanged, the peptide chain comprising the heavychain constant domain 1 CH1 is referred to herein as the “heavy chain”of the (crossover) Fab molecule. Conversely, in a crossover Fab moleculewherein the constant domains of the Fab light chain and the Fab heavychain are exchanged, the peptide chain comprising the heavy chainvariable domain VH is referred to herein as the “heavy chain” of the(crossover) Fab molecule.

In contrast thereto, by a “conventional” Fab molecule is meant a Fabmolecule in its natural format, i.e. comprising a heavy chain composedof the heavy chain variable and constant domains (VH-CH1, in N- toC-terminal direction), and a light chain composed of the light chainvariable and constant domains (VL-CL, in N- to C-terminal direction).

The term “immunoglobulin molecule” refers to a protein having thestructure of a naturally occurring antibody. For example,immunoglobulins of the IgG class are heterotetrameric glycoproteins ofabout 150,000 daltons, composed of two light chains and two heavy chainsthat are disulfide-bonded. From N- to C-terminus, each heavy chain has avariable domain (VH), also called a variable heavy domain or a heavychain variable region, followed by three constant domains (CH1, CH2, andCH3), also called a heavy chain constant region. Similarly, from N- toC-terminus, each light chain has a variable domain (VL), also called avariable light domain or a light chain variable region, followed by aconstant light (CL) domain, also called a light chain constant region.The heavy chain of an immunoglobulin may be assigned to one of fivetypes, called α (IgA), δ (IgD), ε (IgE), γ (IgG), or μ (IgM), some ofwhich may be further divided into subtypes, e.g. γ₁ (IgG₁), γ₂ (IgG₂),γ₃ (IgG₃), γ₄ (IgG₄), α₁ (IgA₁) and α₂ (IgA₂). The light chain of animmunoglobulin may be assigned to one of two types, called kappa (κ) andlambda (λ), based on the amino acid sequence of its constant domain. Animmunoglobulin essentially consists of two Fab molecules and an Fcdomain, linked via the immunoglobulin hinge region.

The term “antibody” herein is used in the broadest sense and encompassesvarious antibody structures, including but not limited to monoclonalantibodies, polyclonal antibodies, multispecific antibodies (e.g.bispecific antibodies), and antibody fragments so long as they exhibitthe desired antigen-binding activity.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies, i.e.the individual antibodies comprised in the population are identicaland/or bind the same epitope, except for possible variant antibodies,e.g., containing naturally occurring mutations or arising duringproduction of a monoclonal antibody preparation, such variants generallybeing present in minor amounts. In contrast to polyclonal antibodypreparations, which typically include different antibodies directedagainst different determinants (epitopes), each monoclonal antibody of amonoclonal antibody preparation is directed against a single determinanton an antigen. Thus, the modifier “monoclonal” indicates the characterof the antibody as being obtained from a substantially homogeneouspopulation of antibodies, and is not to be construed as requiringproduction of the antibody by any particular method. For example, themonoclonal antibodies to be used in accordance with the presentinvention may be made by a variety of techniques, including but notlimited to the hybridoma method, recombinant DNA methods, phage-displaymethods, and methods utilizing transgenic animals containing all or partof the human immunoglobulin loci, such methods and other exemplarymethods for making monoclonal antibodies being described herein.

An “isolated” antibody is one which has been separated from a componentof its natural environment, i.e. that is not in its natural milieu. Noparticular level of purification is required. For example, an isolatedantibody can be removed from its native or natural environment.Recombinantly produced antibodies expressed in host cells are consideredisolated for the purpose of the invention, as are native or recombinantantibodies which have been separated, fractionated, or partially orsubstantially purified by any suitable technique. As such, theantibodies and bispecific antigen binding molecules of the presentinvention are isolated. In some embodiments, an antibody is purified togreater than 95% or 99% purity as determined by, for example,electrophoretic (e.g., SDS-PAGE, isoelectric focusing (IEF), capillaryelectrophoresis) or chromatographic (e.g., ion exchange or reverse phaseHPLC) methods. For review of methods for assessment of antibody purity,see, e.g., Flatman et al., J. Chromatogr. B 848:79-87 (2007).

The terms “full length antibody,” “intact antibody,” and “wholeantibody” are used herein interchangeably to refer to an antibody havinga structure substantially similar to a native antibody structure.

An “antibody fragment” refers to a molecule other than an intactantibody that comprises a portion of an intact antibody that binds theantigen to which the intact antibody binds. Examples of antibodyfragments include but are not limited to Fv, Fab, Fab′, Fab′-SH,F(ab′)₂, diabodies, linear antibodies, single-chain antibody molecules(e.g. scFv), and single-domain antibodies. For a review of certainantibody fragments, see Hudson et al., Nat Med 9, 129-134 (2003). For areview of scFv fragments, see e.g. Plückthun, in The Pharmacology ofMonoclonal Antibodies, vol. 113, Rosenburg and Moore eds.,Springer-Verlag, New York, pp. 269-315 (1994); see also WO 93/16185; andU.S. Pat. Nos. 5,571,894 and 5,587,458. For discussion of Fab andF(ab′)₂ fragments comprising salvage receptor binding epitope residuesand having increased in vivo half-life, see U.S. Pat. No. 5,869,046.Diabodies are antibody fragments with two antigen-binding sites that maybe bivalent or bispecific. See, for example, EP 404,097; WO 1993/01161;Hudson et al., Nat Med 9, 129-134 (2003); and Hollinger et al., ProcNatl Acad Sci USA 90, 6444-6448 (1993). Triabodies and tetrabodies arealso described in Hudson et al., Nat Med 9, 129-134 (2003).Single-domain antibodies are antibody fragments comprising all or aportion of the heavy chain variable domain or all or a portion of thelight chain variable domain of an antibody. In certain embodiments, asingle-domain antibody is a human single-domain antibody (Domantis,Inc., Waltham, Mass.; see e.g. U.S. Pat. No. 6,248,516 B1). Antibodyfragments can be made by various techniques, including but not limitedto proteolytic digestion of an intact antibody as well as production byrecombinant host cells (e.g. E. coli or phage), as described herein.

The term “antigen binding domain” refers to the part of an antibody thatcomprises the area which specifically binds to and is complementary topart or all of an antigen. An antigen binding domain may be provided by,for example, one or more antibody variable domains (also called antibodyvariable regions). Particularly, an antigen binding domain comprises anantibody light chain variable domain (VL) and an antibody heavy chainvariable domain (VH).

The term “variable region” or “variable domain” refers to the domain ofan antibody heavy or light chain that is involved in binding theantibody to antigen. The variable domains of the heavy chain and lightchain (VH and VL, respectively) of a native antibody generally havesimilar structures, with each domain comprising four conserved frameworkregions (FRs) and three hypervariable regions (HVRs). See, e.g., Kindtet al., Kuby Immunology, 6th ed., W.H. Freeman and Co., page 91 (2007).A single VH or VL domain may be sufficient to confer antigen-bindingspecificity. As used herein in connection with variable regionsequences, “Kabat numbering” refers to the numbering system set forth byKabat et al., Sequences of Proteins of Immunological Interest, 5th Ed.Public Health Service, National Institutes of Health, Bethesda, Md.(1991).

As used herein, the amino acid positions of all constant regions anddomains of the heavy and light chain are numbered according to the Kabatnumbering system described in Kabat, et al., Sequences of Proteins ofImmunological Interest, 5th ed., Public Health Service, NationalInstitutes of Health, Bethesda, Md. (1991), referred to as “numberingaccording to Kabat” or “Kabat numbering” herein. Specifically the Kabatnumbering system (see pages 647-660 of Kabat, et al., Sequences ofProteins of Immunological Interest, 5th ed., Public Health Service,National Institutes of Health, Bethesda, Md. (1991)) is used for thelight chain constant domain CL of kappa and lambda isotype and the KabatEU index numbering system (see pages 661-723) is used for the heavychain constant domains (CH1, Hinge, CH2 and CH3), which is hereinfurther clarified by referring to “numbering according to Kabat EUindex” in this case.

The term “hypervariable region” or “HVR”, as used herein, refers to eachof the regions of an antibody variable domain which are hypervariable insequence (“complementarity determining regions” or “CDRs”) and/or formstructurally defined loops (“hypervariable loops”) and/or contain theantigen-contacting residues (“antigen contacts”). Generally, antibodiescomprise six HVRs; three in the VH (H1, H2, H3), and three in the VL(L1, L2, L3). Exemplary HVRs herein include:

-   -   (a) hypervariable loops occurring at amino acid residues 26-32        (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101        (H3) (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987));    -   (b) CDRs occurring at amino acid residues 24-34 (L1), 50-56        (L2), 89-97 (L3), 31-35b (H1), 50-65 (H2), and 95-102 (H3)        (Kabat et al., Sequences of Proteins of Immunological Interest,        5th Ed. Public Health Service, National Institutes of Health,        Bethesda, Md. (1991));    -   (c) antigen contacts occurring at amino acid residues 27c-36        (L1), 46-55 (L2), 89-96 (L3), 30-35b (H1), 47-58 (H2), and        93-101 (H3) (MacCallum et al. J. Mol. Biol. 262: 732-745        (1996)); and    -   (d) combinations of (a), (b), and/or (c), including HVR amino        acid residues 46-56 (L2), 47-56 (L2), 48-56 (L2), 49-56 (L2),        26-35 (H1), 26-35b (H1), 49-65 (H2), 93-102 (H3), and 94-102        (H3).

Unless otherwise indicated, HVR residues and other residues in thevariable domain (e.g., FR residues) are numbered herein according toKabat et al., supra.

“Framework” or “FR” refers to variable domain residues other thanhypervariable region (HVR) residues. The FR of a variable domaingenerally consists of four FR domains: FR1, FR2, FR3, and FR4.Accordingly, the HVR and FR sequences generally appear in the followingorder in VH (or VL): FR1-H1 (L1)-FR2-H2(L2)-FR3-H3 (L3)-FR4.

A “humanized” antibody refers to a chimeric antibody comprising aminoacid residues from non-human HVRs and amino acid residues from humanFRs. In certain embodiments, a humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the HVRs (e.g., CDRs) correspond tothose of a non-human antibody, and all or substantially all of the FRscorrespond to those of a human antibody. Such variable domains arereferred to herein as “humanized variable region”. A humanized antibodyoptionally may comprise at least a portion of an antibody constantregion derived from a human antibody. In some embodiments, some FRresidues in a humanized antibody are substituted with correspondingresidues from a non-human antibody (e.g., the antibody from which theHVR residues are derived), e.g., to restore or improve antibodyspecificity or affinity. A “humanized form” of an antibody, e.g. of anon-human antibody, refers to an antibody that has undergonehumanization. Other forms of “humanized antibodies” encompassed by thepresent invention are those in which the constant region has beenadditionally modified or changed from that of the original antibody togenerate the properties according to the invention, especially in regardto C1q binding and/or Fc receptor (FcR) binding.

A “human antibody” is one which possesses an amino acid sequence whichcorresponds to that of an antibody produced by a human or a human cellor derived from a non-human source that utilizes human antibodyrepertoires or other human antibody-encoding sequences. This definitionof a human antibody specifically excludes a humanized antibodycomprising non-human antigen-binding residues. In certain embodiments, ahuman antibody is derived from a non-human transgenic mammal, forexample a mouse, a rat, or a rabbit. In certain embodiments, a humanantibody is derived from a hybridoma cell line. Antibodies or antibodyfragments isolated from human antibody libraries are also consideredhuman antibodies or human antibody fragments herein.

The “class” of an antibody or immunoglobulin refers to the type ofconstant domain or constant region possessed by its heavy chain. Thereare five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, andseveral of these may be further divided into subclasses (isotypes),e.g., IgG₁, IgG₂, IgG₃, IgG₄, IgA₁, and IgA₂. The heavy chain constantdomains that correspond to the different classes of immunoglobulins arecalled α, δ, ε, γ, and μ, respectively.

The term “Fc domain” or “Fc region” herein is used to define aC-terminal region of an immunoglobulin heavy chain that contains atleast a portion of the constant region. The term includes nativesequence Fc regions and variant Fc regions. Although the boundaries ofthe Fc region of an IgG heavy chain might vary slightly, the human IgGheavy chain Fc region is usually defined to extend from Cys226, or fromPro230, to the carboxyl-terminus of the heavy chain. However, antibodiesproduced by host cells may undergo post-translational cleavage of one ormore, particularly one or two, amino acids from the C-terminus of theheavy chain. Therefore an antibody produced by a host cell by expressionof a specific nucleic acid molecule encoding a full-length heavy chainmay include the full-length heavy chain, or it may include a cleavedvariant of the full-length heavy chain (also referred to herein as a“cleaved variant heavy chain”). This may be the case where the final twoC-terminal amino acids of the heavy chain are glycine (G446) and lysine(K447, numbering according to Kabat EU index). Therefore, the C-terminallysine (Lys447), or the C-terminal glycine (Gly446) and lysine (K447),of the Fc region may or may not be present. Amino acid sequences ofheavy chains including Fc domains (or a subunit of an Fc domain asdefined herein) are denoted herein without C-terminal glycine-lysinedipeptide if not indicated otherwise. In one embodiment of theinvention, a heavy chain including a subunit of an Fc domain asspecified herein, comprised in an antibody or bispecific antigen bindingmolecule according to the invention, comprises an additional C-terminalglycine-lysine dipeptide (G446 and K447, numbering according to EU indexof Kabat). In one embodiment of the invention, a heavy chain including asubunit of an Fc domain as specified herein, comprised in an antibody orbispecific antigen binding molecule according to the invention,comprises an additional C-terminal glycine residue (G446, numberingaccording to EU index of Kabat). Compositions of the invention, such asthe pharmaceutical compositions described herein, comprise a populationof antibodies or bispecific antigen binding molecules of the invention.The population of antibodies or bispecific antigen binding molecules maycomprise molecules having a full-length heavy chain and molecules havinga cleaved variant heavy chain. The population of antibodies orbispecific antigen binding molecules may consist of a mixture ofmolecules having a full-length heavy chain and molecules having acleaved variant heavy chain, wherein at least 50%, at least 60%, atleast 70%, at least 80% or at least 90% of the antibodies or bispecificantigen binding molecules have a cleaved variant heavy chain. In oneembodiment of the invention a composition comprising a population ofantibodies or bispecific antigen binding molecules of the inventioncomprises an antibody or bispecific antigen binding molecule comprisinga heavy chain including a subunit of an Fc domain as specified hereinwith an additional C-terminal glycine-lysine dipeptide (G446 and K447,numbering according to EU index of Kabat). In one embodiment of theinvention a composition comprising a population of antibodies orbispecific antigen binding molecules of the invention comprises anantibody or bispecific antigen binding molecule comprising a heavy chainincluding a subunit of an Fc domain as specified herein with anadditional C-terminal glycine residue (G446, numbering according to EUindex of Kabat). In one embodiment of the invention such a compositioncomprises a population of antibodies or bispecific antigen bindingmolecules comprised of molecules comprising a heavy chain including asubunit of an Fc domain as specified herein; molecules comprising aheavy chain including a subunit of a Fc domain as specified herein withan additional C-terminal glycine residue (G446, numbering according toEU index of Kabat); and molecules comprising a heavy chain including asubunit of an Fc domain as specified herein with an additionalC-terminal glycine-lysine dipeptide (G446 and K447, numbering accordingto EU index of Kabat). Unless otherwise specified herein, numbering ofamino acid residues in the Fc region or constant region is according tothe EU numbering system, also called the EU index, as described in Kabatet al., Sequences of Proteins of Immunological Interest, 5th Ed. PublicHealth Service, National Institutes of Health, Bethesda, Md., 1991 (seealso above). A “subunit” of an Fc domain as used herein refers to one ofthe two polypeptides forming the dimeric Fc domain, i.e. a polypeptidecomprising C-terminal constant regions of an immunoglobulin heavy chain,capable of stable self-association. For example, a subunit of an IgG Fcdomain comprises an IgG CH2 and an IgG CH3 constant domain.

A “modification promoting the association of the first and the secondsubunit of the Fc domain” is a manipulation of the peptide backbone orthe post-translational modifications of an Fc domain subunit thatreduces or prevents the association of a polypeptide comprising the Fcdomain subunit with an identical polypeptide to form a homodimer. Amodification promoting association as used herein particularly includesseparate modifications made to each of the two Fc domain subunitsdesired to associate (i.e. the first and the second subunit of the Fcdomain), wherein the modifications are complementary to each other so asto promote association of the two Fc domain subunits. For example, amodification promoting association may alter the structure or charge ofone or both of the Fc domain subunits so as to make their associationsterically or electrostatically favorable, respectively. Thus,(hetero)dimerization occurs between a polypeptide comprising the firstFc domain subunit and a polypeptide comprising the second Fc domainsubunit, which might be non-identical in the sense that furthercomponents fused to each of the subunits (e.g. antigen binding moieties)are not the same. In some embodiments the modification promotingassociation comprises an amino acid mutation in the Fc domain,specifically an amino acid substitution. In a particular embodiment, themodification promoting association comprises a separate amino acidmutation, specifically an amino acid substitution, in each of the twosubunits of the Fc domain.

The term “effector functions” refers to those biological activitiesattributable to the Fc region of an antibody, which vary with theantibody isotype. Examples of antibody effector functions include: C1qbinding and complement dependent cytotoxicity (CDC), Fc receptorbinding, antibody-dependent cell-mediated cytotoxicity (ADCC),antibody-dependent cellular phagocytosis (ADCP), cytokine secretion,immune complex-mediated antigen uptake by antigen presenting cells, downregulation of cell surface receptors (e.g. B cell receptor), and B cellactivation.

As used herein, the terms “engineer, engineered, engineering”, areconsidered to include any manipulation of the peptide backbone or thepost-translational modifications of a naturally occurring or recombinantpolypeptide or fragment thereof. Engineering includes modifications ofthe amino acid sequence, of the glycosylation pattern, or of the sidechain group of individual amino acids, as well as combinations of theseapproaches.

The term “amino acid mutation” as used herein is meant to encompassamino acid substitutions, deletions, insertions, and modifications. Anycombination of substitution, deletion, insertion, and modification canbe made to arrive at the final construct, provided that the finalconstruct possesses the desired characteristics, e.g., reduced bindingto an Fc receptor, or increased association with another peptide. Aminoacid sequence deletions and insertions include amino- and/orcarboxy-terminal deletions and insertions of amino acids. Particularamino acid mutations are amino acid substitutions. For the purpose ofaltering e.g. the binding characteristics of an Fc region,non-conservative amino acid substitutions, i.e. replacing one amino acidwith another amino acid having different structural and/or chemicalproperties, are particularly preferred. Amino acid substitutions includereplacement by non-naturally occurring amino acids or by naturallyoccurring amino acid derivatives of the twenty standard amino acids(e.g. 4-hydroxyproline, 3-methylhistidine, ornithine, homoserine,5-hydroxylysine). Amino acid mutations can be generated using genetic orchemical methods well known in the art. Genetic methods may includesite-directed mutagenesis, PCR, gene synthesis and the like. It iscontemplated that methods of altering the side chain group of an aminoacid by methods other than genetic engineering, such as chemicalmodification, may also be useful. Various designations may be usedherein to indicate the same amino acid mutation. For example, asubstitution from proline at position 329 of the Fc domain to glycinecan be indicated as 329G, G329, G329, P329G, or Pro329Gly.

“Percent (%) amino acid sequence identity” with respect to a referencepolypeptide sequence is defined as the percentage of amino acid residuesin a candidate sequence that are identical with the amino acid residuesin the reference polypeptide sequence, after aligning the sequences andintroducing gaps, if necessary, to achieve the maximum percent sequenceidentity, and not considering any conservative substitutions as part ofthe sequence identity. Alignment for purposes of determining percentamino acid sequence identity can be achieved in various ways that arewithin the skill in the art, for instance, using publicly availablecomputer software such as BLAST, BLAST-2, Clustal W, Megalign (DNASTAR)software or the FASTA program package. Those skilled in the art candetermine appropriate parameters for aligning sequences, including anyalgorithms needed to achieve maximal alignment over the full length ofthe sequences being compared. For purposes herein, however, % amino acidsequence identity values are generated using the ggsearch program of theFASTA package version 36.3.8c or later with a BLOSUM50 comparisonmatrix. The FASTA program package was authored by W. R. Pearson and D.J. Lipman (1988), “Improved Tools for Biological Sequence Analysis”,PNAS 85:2444-2448; W. R. Pearson (1996) “Effective protein sequencecomparison” Meth. Enzymol. 266:227-258; and Pearson et. al. (1997)Genomics 46:24-36, and is publicly available fromhttp://fasta.bioch.virginia.edu/fasta_www2/fasta_down.shtml.Alternatively, a public server accessible athttp://fasta.bioch.virginia.edu/fasta_www2/index.cgi can be used tocompare the sequences, using the ggsearch (global protein:protein)program and default options (BLOSUM50; open: −10; ext: −2; Ktup=2) toensure a global, rather than local, alignment is performed. Percentamino acid identity is given in the output alignment header.

The term “polynucleotide” refers to an isolated nucleic acid molecule orconstruct, e.g. messenger RNA (mRNA), virally-derived RNA, or plasmidDNA (pDNA). A polynucleotide may comprise a conventional phosphodiesterbond or a non-conventional bond (e.g. an amide bond, such as found inpeptide nucleic acids (PNA). The term “nucleic acid molecule” refers toany one or more nucleic acid segments, e.g. DNA or RNA fragments,present in a polynucleotide.

By “isolated” nucleic acid molecule or polynucleotide is intended anucleic acid molecule, DNA or RNA, which has been removed from itsnative environment. For example, a recombinant polynucleotide encoding apolypeptide contained in a vector is considered isolated for thepurposes of the present invention. Further examples of an isolatedpolynucleotide include recombinant polynucleotides maintained inheterologous host cells or purified (partially or substantially)polynucleotides in solution. An isolated polynucleotide includes apolynucleotide molecule contained in cells that ordinarily contain thepolynucleotide molecule, but the polynucleotide molecule is presentextrachromosomally or at a chromosomal location that is different fromits natural chromosomal location. Isolated RNA molecules include in vivoor in vitro RNA transcripts of the present invention, as well aspositive and negative strand forms, and double-stranded forms. Isolatedpolynucleotides or nucleic acids according to the present inventionfurther include such molecules produced synthetically. In addition, apolynucleotide or a nucleic acid may be or may include a regulatoryelement such as a promoter, ribosome binding site, or a transcriptionterminator.

“Isolated polynucleotide (or nucleic acid) encoding [e.g. an antibody orbispecific antigen binding molecule of the invention]” refers to one ormore polynucleotide molecules encoding antibody heavy and light chains(or fragments thereof), including such polynucleotide molecule(s) in asingle vector or separate vectors, and such nucleic acid molecule(s)present at one or more locations in a host cell.

The term “expression cassette” refers to a polynucleotide generatedrecombinantly or synthetically, with a series of specified nucleic acidelements that permit transcription of a particular nucleic acid in atarget cell. The recombinant expression cassette can be incorporatedinto a plasmid, chromosome, mitochondrial DNA, plastid DNA, virus, ornucleic acid fragment. Typically, the recombinant expression cassetteportion of an expression vector includes, among other sequences, anucleic acid sequence to be transcribed and a promoter. In certainembodiments, the expression cassette comprises polynucleotide sequencesthat encode antibodies or bispecific antigen binding molecules of theinvention or fragments thereof.

The term “vector” or “expression vector” refers to a DNA molecule thatis used to introduce and direct the expression of a specific gene towhich it is operably associated in a cell. The term includes the vectoras a self-replicating nucleic acid structure as well as the vectorincorporated into the genome of a host cell into which it has beenintroduced. The expression vector of the present invention comprises anexpression cassette. Expression vectors allow transcription of largeamounts of stable mRNA. Once the expression vector is inside the cell,the ribonucleic acid molecule or protein that is encoded by the gene isproduced by the cellular transcription and/or translation machinery. Inone embodiment, the expression vector of the invention comprises anexpression cassette that comprises polynucleotide sequences that encodeantibodies or bispecific antigen binding molecules of the invention orfragments thereof.

The terms “host cell”, “host cell line,” and “host cell culture” areused interchangeably and refer to cells into which exogenous nucleicacid has been introduced, including the progeny of such cells. Hostcells include “transformants” and “transformed cells,” which include theprimary transformed cell and progeny derived therefrom without regard tothe number of passages. Progeny may not be completely identical innucleic acid content to a parent cell, but may contain mutations. Mutantprogeny that have the same function or biological activity as screenedor selected for in the originally transformed cell are included herein.A host cell is any type of cellular system that can be used to generatethe antibodies or bispecific antigen binding molecules of the presentinvention. Host cells include cultured cells, e.g. mammalian culturedcells, such as HEK cells, CHO cells, BHK cells, NS0 cells, SP2/0 cells,YO myeloma cells, P3X63 mouse myeloma cells, PER cells, PER.C6 cells orhybridoma cells, yeast cells, insect cells, and plant cells, to nameonly a few, but also cells comprised within a transgenic animal,transgenic plant or cultured plant or animal tissue. An “activating Fcreceptor” is an Fc receptor that following engagement by an Fc domain ofan antibody elicits signaling events that stimulate the receptor-bearingcell to perform effector functions. Human activating Fc receptorsinclude FcγRIIIa (CD16a), FcγRI (CD64), FcγRIIa (CD32), and FcαRI(CD89).

Antibody-dependent cell-mediated cytotoxicity (ADCC) is an immunemechanism leading to the lysis of antibody-coated target cells by immuneeffector cells. The target cells are cells to which antibodies orderivatives thereof comprising an Fc region specifically bind, generallyvia the protein part that is N-terminal to the Fc region. As usedherein, the term “reduced ADCC” is defined as either a reduction in thenumber of target cells that are lysed in a given time, at a givenconcentration of antibody in the medium surrounding the target cells, bythe mechanism of ADCC defined above, and/or an increase in theconcentration of antibody in the medium surrounding the target cells,required to achieve the lysis of a given number of target cells in agiven time, by the mechanism of ADCC. The reduction in ADCC is relativeto the ADCC mediated by the same antibody produced by the same type ofhost cells, using the same standard production, purification,formulation and storage methods (which are known to those skilled in theart), but that has not been engineered. For example the reduction inADCC mediated by an antibody comprising in its Fc domain an amino acidsubstitution that reduces ADCC, is relative to the ADCC mediated by thesame antibody without this amino acid substitution in the Fc domain.Suitable assays to measure ADCC are well known in the art (see e.g. PCTpublication no. WO 2006/082515 or PCT publication no. WO 2012/130831).

An “effective amount” of an agent refers to the amount that is necessaryto result in a physiological change in the cell or tissue to which it isadministered.

A “therapeutically effective amount” of an agent, e.g. a pharmaceuticalcomposition, refers to an amount effective, at dosages and for periodsof time necessary, to achieve the desired therapeutic or prophylacticresult. A therapeutically effective amount of an agent for exampleeliminates, decreases, delays, minimizes or prevents adverse effects ofa disease.

An “individual” or “subject” is a mammal. Mammals include, but are notlimited to, domesticated animals (e.g. cows, sheep, cats, dogs, andhorses), primates (e.g. humans and non-human primates such as monkeys),rabbits, and rodents (e.g. mice and rats). Particularly, the individualor subject is a human.

The term “pharmaceutical composition” refers to a preparation which isin such form as to permit the biological activity of an activeingredient contained therein to be effective, and which contains noadditional components which are unacceptably toxic to a subject to whichthe composition would be administered.

A “pharmaceutically acceptable carrier” refers to an ingredient in apharmaceutical composition, other than an active ingredient, which isnontoxic to a subject. A pharmaceutically acceptable carrier includes,but is not limited to, a buffer, excipient, stabilizer, or preservative.

As used herein, “treatment” (and grammatical variations thereof such as“treat” or “treating”) refers to clinical intervention in an attempt toalter the natural course of a disease in the individual being treated,and can be performed either for prophylaxis or during the course ofclinical pathology. Desirable effects of treatment include, but are notlimited to, preventing occurrence or recurrence of disease, alleviationof symptoms, diminishment of any direct or indirect pathologicalconsequences of the disease, preventing metastasis, decreasing the rateof disease progression, amelioration or palliation of the disease state,and remission or improved prognosis. In some embodiments, antibodies orbispecific antigen binding molecules of the invention are used to delaydevelopment of a disease or to slow the progression of a disease.

The term “package insert” is used to refer to instructions customarilyincluded in commercial packages of therapeutic products, that containinformation about the indications, usage, dosage, administration,combination therapy, contraindications and/or warnings concerning theuse of such therapeutic products.

Detailed Description of the Embodiments

The invention provides antibodies and bispecific antigen bindingmolecules that bind HLA-A2/WT1, particularly HLA-A2/WT1_(RMF) orHLA-A2/WT1_(VLD), most particularly HLA-A2/WT1_(RMF), and have goodaffinity and specificity as required for therapeutic purposes. Inaddition, the molecules have also other favorable properties fortherapeutic application, e.g. with respect to efficacy and/or safety aswell as produceability.

HLA-A2/WT1 Antibody

The present inventors have developed novel antibodies that bind toHLA-A2/WT1 with particularly good affinity and specificity. Forinstance, as shown in the Examples, the inventors have developedantibodies that are remarkably selective for HLA-A2/WT1 (specificallyHLA-A2/WT1_(RMF)) over complexes of HLA-A2 with other, structurallysimilar peptides.

Thus, in certain aspects, the invention provides an antibody that bindsto HLA-A2/WT1 and has any of the following features.

In one embodiment, the antibody has a monovalent affinity to HLA-A2/WT1with a dissociation constant (K_(D)) of lower than about 100 nM, lowerthan about 75 nM, or lower than about 50 nM. In one embodiment, theantibody has a bivalent affinity (avidity) to HLA-A2/WT1 with anapparent K_(D) of lower than about 1.5 nM, lower than about 1 nM, orlower than about 0.75 nM. In one embodiment, the bivalent affinity(avidity) of the antibody is at 10-fold, at least 20-fold, at least50-fold, or at least 100-fold higher than the monovalent affinity of theantibody in terms of (apparent) K_(D).

In one embodiment, the affinity is determined by Surface PlasmonResonance (SPR) at 25° C. In one embodiment, the monovalent affinity isdetermined in a Fab molecule format of the antibody.

In one embodiment, the bivalent affinity (avidity) is determined in anIgG molecule format of the antibody.

In a specific embodiment, the affinity of the antibody is determined asfollows:

Experiments are performed at 25° C. using PBST as running buffer (10 mMPBS, pH 7.4 and 0.005% (v/v) Tween 20). A ProteOn XPR36 biosensorequipped with GLC and GLM sensor chips and coupling reagents (10 mMsodium acetate pH 4.5, sulfo-N-hydroxysuccinimide [sulfo-NHS],1-ethyl-3-(3-dimethylaminpropyl)-carbodiimide hydrochloride [EDC] andethanolamine) from BioRad Inc. (Hercules, Calif.) is used.Immobilizations are performed at 30 μl/min on a GLM chip. pAb (goat)anti human IgG, F(ab)₂ specific antibody (Jackson ImmunoResearch) iscoupled in vertical direction using a standard amine-coupling procedure:all six ligand channels are activated for 5 min with a mixture of EDC(200 mM) and sulfo-NHS (50 mM). Immediately after the surfaces areactivated, pAb (goat) anti human IgG, F(ab)₂ specific antibody (50μg/ml, 10 mM sodium acetate, pH 5) is injected across all six channelsfor 5 min. Finally, channels are blocked with a 5 min injection of 1 Methanolamine-HCl (pH 8.5). The Fab variants are captured from E. colisupernatants by simultaneous injection along five of the separatehorizontal channels (30 μl/min) for 5 min. Conditioned medium isinjected along the sixth channel to provide an ‘in-line’ blank fordouble referencing purposes. One-shot kinetic measurements are performedby injection of a dilution series of HLA-WT1 (e.g. HLA-A2/WT1_(RMF) orHLA-A2/WT1_(VLD)) (100, 50, 25, 12.5, 6.25, 0 nM, 50 μl/min) for 2 minalong the vertical channels. Dissociation is monitored for 3 min.Kinetic data is analyzed in ProteOn Manager v. 2.1. Processing of thereaction spot data involves applying an interspot-reference and adouble-reference step using an inline buffer blank (Myszka, J MolRecognit (1999) 12, 279-284). The processed data from replicate one-shotinjections are fit to a simple 1:1 Langmuir binding model without masstransport (O'Shannessy et al., Anal Biochem (1993) 212, 457-468).

The antibody of the invention specifically binds to HLA-A2/WT1 (i.e. acomplex of a HLA-A2 molecule and a WT1-derived peptide). In someembodiments, the antibody of the invention specifically binds toHLA-A2/WT1_(RMF) (i.e. a complex of a HLA-A2 molecule and the WT1_(RMF)peptide). In a more specific embodiment, the antibody specifically bindsto HLA-A201/WT1_(RMF) (i.e. a complex of a HLA-A201 molecule and theWT1_(RMF) peptide). Antibodies of the invention that bind toHLA-A2/WT1_(RMF) include antibodies 11D06, 33H09 and 5E11 describedherein. In other embodiments, the antibody of the invention specificallybinds to HLA-A2/WT1_(VLD) (i.e. a complex of a HLA-A2 molecule and theWT1_(VLD) peptide). In a more specific embodiment, the antibodyspecifically binds to HLA-A201/WT1_(VLD) (i.e. a complex of a HLA-A201molecule and the WT1_(VLD)) peptide). Antibodies of the invention thatbind to HLA-A2/WT1_(VLD) include antibodies 11B09, 13B04 and 5C01described herein.

In one embodiment, specific binding of the antibody is determined byflow cytometry using HLA-A2/peptide (e.g. WT1_(RMF) or WT1_(VLD)peptide)-expressing cells, particularly peptide-pulsed T2 cells.

In a specific embodiment, specific binding of the antibody is determinedas follows: T2 cells are prepared as a cell suspension at 10⁶ cells/mlin IMDM medium (Gibco by Life Technologies, Cat No. 31980-048),supplemented with 10% FBS (Gibco, Cat No. 16140-071)+1%Penicillin-Streptomycin (Gibco, Cat No. 15070-063) (complete medium).Cells are kept in a total volume of 10 ml in a tube, and incubated with10 μl of peptide (e.g. WT1_(VLD) peptide (SEQ ID NO: 77), or RMF peptide(SEQ ID NO: 78)) at 10⁻² M (final concentration of the peptide: 10⁻⁵M)for 2 hours at 37° C. with 5% CO₂. After washing, cells are suspended incold PBS and incubated with titrated concentration of antibody in IgGformat (e.g. 10 μg/ml to 0.00064 μg/ml) for 1 hour at 4° C., followed byincubation with a secondary anti-human IgG-Fc phycoerythrin(PE)-conjugated antibody (Jackson Laboratories, Cat No. 109-116-098) for30 min. Cells are acquired on FACS LSR II (BD), and data are presentedas mean fluorescence intensity (MFI) of PE in Graphpad Prism.

In one embodiment, the antibody of the invention does not significantlybind to HLA-A2 alone (i.e. without a peptide) or HLA-A2 with a peptideother than a WT1-derived peptide such as WT1_(RMF) or WT1_(VLD)).

In one embodiment, the antibody does not significantly bind to HLA-A2 inthe absence of a WT1-derived peptide, particularly WT1R1 or WT1_(VLD).In one embodiment, the antibody binds to HLA-A2/WT1 (specificallyHLA-A2/WT1_(RMF) or HLA-A2/WT1_(VLD)) with an EC50 that is at least 5,at least 10, at least 15, at least 20, at least 25, at least 50, atleast 75 or at least 100 times lower than the EC50 for binding to HLA-A2in the absence of a WT1-derived peptide (specifically WT1_(RMF) or WT Inone embodiment, the antibody binds to HLA-A2/WT1 (specificallyHLA-A2/WT1_(RMF) or HLA-A2/WT1_(VLD)), but does not significantly bindto HLA-A2 with a peptide selected from the peptides in Table 5 (thepeptides of SEQ ID NOs 79-105). In one embodiment, the antibody binds toHLA-A2/WT1 (specifically HLA-A2/WT1_(RMF) or HLA-A2/WT1_(VLD)), but doesnot significantly bind to HLA-A2 with any of the peptides in Table 5(the peptides of SEQ ID NOs 79-105).

In one embodiment, the antibody binds to HLA-A2/WT1 (specificallyHLA-A2/WT1_(RMF) or HLA-A2/WT1_(VLD)) with an EC50 that is at least 5,at least 10, at least 15, at least 20, at least 25, at least 50, atleast 75 or at least 100 times lower than the EC50 for binding to HLA-A2with a peptide selected from the peptides in Table 5 (the peptides ofSEQ ID NOs 79-105). In one embodiment, the antibody binds to HLA-A2/WT1(specifically HLA-A2/WT1_(RMF) or HLA-A2/WT1_(VLD)) with an EC50 that isat least 5, at least 10, at least 15, at least 20, at least 25, at least50, at least 75 or at least 100 times lower than the EC50 for binding toHLA-A2 with any of the peptides in Table 5 (the peptides of SEQ ID NOs79-105).

In one embodiment, the EC50 is determined flow cytometry usingHLA-A2/peptide (e.g. WT1_(RMF) or WT1_(VLD) peptide)-expressing cells,particularly peptide-pulsed T2 cells.

In a specific embodiment, the EC50 is determined as follows:

T2 cells (ATCC, Cat. No. CRL-1992) are prepared as a cell suspension at10⁶ cells/ml in IMDM medium (Gibco by Life Technologies, Cat No.31980-048), supplemented with 10% FBS (Gibco, Cat No. 16140-071)+1%Penicillin-Streptomycin (Gibco, Cat No. 15070-063) (complete medium).Cells are kept in a total volume of 10 ml in a tube, and incubated with10 μl of peptide (e.g. WT1_(VLD) peptide (SEQ ID NO: 77), or RMF peptide(SEQ ID NO: 78)) at 10⁻² M (final concentration of the peptide: 10⁻⁵M)for 2 hours at 37° C. with 5% CO₂. After washing, cells are suspended incold PBS and incubated with titrated concentration of antibody in IgGformat (e.g. 10 μg/ml to 0.00064 μg/ml) for 1 hour at 4° C., followed byincubation with a secondary anti-human IgG-Fc phycoerythrin(PE)-conjugated antibody (Jackson Laboratories, Cat No. 109-116-098) for30 min. Cells are acquired on FACS LSR II (BD), and data are presentedas mean fluorescence intensity (MFI) of PE in Graphpad Prism. EC₅₀values are calculated in Microsoft® Excel using the XLfit® add-on (IDBusiness Solutions, Guildford, UK).

In one aspect, the invention provides an antibody that competes forbinding to HLA-A2/WT1, particularly HLA-A201/WT1_(RMF), with an antibodycomprising a heavy chain variable region (VH) sequence of SEQ ID NO: 7,and a light chain variable region (VL) sequence of SEQ ID NO: 8.

Competition assays may be used to identify an antibody that competeswith the antibody comprising the VH sequence of SEQ ID NO: 7 and the VLsequence of SEQ ID NO: 8 (the reference antibody) for binding toHLA-A2/WT1. In certain embodiments, such a competing antibody binds tothe same epitope (e.g., a linear or a conformational epitope) that isbound by the reference antibody. Detailed exemplary methods for mappingan epitope to which an antibody binds are provided in Morris (1996)“Epitope Mapping Protocols”, in Methods in Molecular Biology vol. 66(Humana Press, Totowa, N.J.) and are also described in the Examplesherein. In an exemplary competition assay, immobilized HLA-A2/WT1 isincubated in a solution comprising a first labeled antibody that bindsto HLA-A2/WT1 (e.g., the antibody comprising the VH sequence of SEQ IDNO: 7 and the VL sequence of SEQ ID NO: 8) and a second unlabeledantibody that is being tested for its ability to compete with the firstantibody for binding to HLA-A2/WT1. The second antibody may be presentin a hybridoma supernatant. As a control, immobilized HLA-A2/WT1 isincubated in a solution comprising the first labeled antibody but notthe second unlabeled antibody. After incubation under conditionspermissive for binding of the first antibody to HLA-A2/WT1, excessunbound antibody is removed, and the amount of label associated withimmobilized HLA-A2/WT1 is measured. If the amount of label associatedwith immobilized HLA-A2/WT1 is substantially reduced in the test samplerelative to the control sample, then that indicates that the secondantibody is competing with the first antibody for binding to HLA-A2/WT1.See Harlow and Lane (1988) Antibodies: A Laboratory Manual ch. 14 (ColdSpring Harbor Laboratory, Cold Spring Harbor, N.Y.).

In one aspect, the invention provides an antibody that binds to the sameepitope of HLA-A2/WT1, particularly HLA-A2/WT1_(RMF), as an antibodycomprising a heavy chain variable region (VH) sequence of SEQ ID NO: 7,and a light chain variable region (VL) sequence of SEQ ID NO: 8.

Screening for antibodies binding to a particular epitope (i.e., thosebinding to the same epitope) can be done using methods routine in theart such as, e.g., without limitation, alanine scanning, peptide blots(see Meth. Mol. Biol. 248 (2004) 443-463), peptide cleavage analysis,epitope excision, epitope extraction, chemical modification of antigens(see Prot. Sci. 9 (2000) 487-496), and cross-blocking (see “Antibodies”,Harlow and Lane (Cold Spring Harbor Press, Cold Spring Harb., N.Y.).

Antigen Structure-based Antibody Profiling (ASAP), also known asModification-Assisted Profiling (MAP), allows to bin a multitude ofmonoclonal antibodies specifically binding to HLA-A2/WT1 based on thebinding profile of each of the antibodies from the multitude tochemically or enzymatically modified antigen surfaces (see, e.g., US2004/0101920). The antibodies in each bin bind to the same epitope whichmay be a unique epitope either distinctly different from or partiallyoverlapping with epitope represented by another bin.

Also competitive binding can be used to easily determine whether anantibody binds to the same epitope of HLA-A2/WT1 as, or competes forbinding with, a reference anti HLA-A2/WT1 antibody. For example, an“antibody that binds to the same epitope” as a reference antibody refersto an antibody that blocks binding of the reference antibody to itsantigen in a competition assay by 50% or more, and conversely, thereference antibody blocks binding of the antibody to its antigen in acompetition assay by 50% or more. Also for example, to determine if anantibody binds to the same epitope of HLA-A2/WT1 as a referenceantibody, the reference antibody is allowed to bind to HLA-A2/WT1 undersaturating conditions. After removal of the excess of the referenceantibody, the ability of an anti HLA-A2/WT1 antibody in question to bindto HLA-A2/WT1 is assessed. If the antibody is able to bind to HLA-A2/WT1after saturation binding of the reference antibody, it can be concludedthat the antibody in question binds to a different epitope than thereference antibody. But, if the antibody in question is not able to bindto HLA-A2/WT1 after saturation binding of the reference antibody, thenthe antibody in question may bind to the same epitope as the epitopebound by the reference antibody. To confirm whether the antibody inquestion binds to the same epitope or is just hampered from binding bysteric reasons routine experimentation can be used (e.g., peptidemutation and binding analyses using ELISA, RIA, surface plasmonresonance, flow cytometry or any other quantitative or qualitativeantibody-binding assay available in the art). This assay should becarried out in two set-ups, i.e. with both of the antibodies being thesaturating antibody. If, in both set-ups, only the first (saturating)antibody is capable of binding to HLA-A2/WT1, then it can be concludedthat the anti-HLA-A2/WT1 antibody in question and the referenceanti-HLA-A2/WT1 antibody compete for binding to HLA-A2/WT1.

In some embodiments two antibodies are deemed to bind to the same or anoverlapping epitope if a 1-, 5-, 10-, 20- or 100-fold excess of oneantibody inhibits binding of the other by at least 50%, at least 75%, atleast 90% or even 99% or more as measured in a competitive binding assay(see, e.g., Junghans et al., Cancer Res. 50 (1990) 1495-1502).

In some embodiments two antibodies are deemed to bind to the sameepitope if essentially all amino acid mutations in the antigen thatreduce or eliminate binding of one antibody also reduce or eliminatebinding of the other. Two antibodies are deemed to have “overlappingepitopes” if only a subset of the amino acid mutations that reduce oreliminate binding of one antibody reduce or eliminate binding of theother.

In one aspect, the invention provides an antibody that binds toHLA-A2/WT1, wherein the antibody binds to an epitope of HLA-A2/WT1,particularly HLA-A2/WT1_(RMF), according to any of the followingembodiments. The epitope may particularly be determined by crystalstructure analysis.

In one embodiment, said epitope comprises at least three amino acidresidues of the WT1 peptide, particularly the WT1_(RMF) peptide shown inSEQ ID NO: 78.

In one embodiment, said epitope comprises at least four amino acidresidues of the WT1 peptide, particularly the WT1_(RMF) peptide shown inSEQ ID NO: 78.

In one embodiment, said epitope comprises at least five amino acidresidues of the WT1 peptide, particularly the WT1_(RMF) peptide shown inSEQ ID NO: 78.

In one embodiment, said epitope comprises at least six amino acidresidues of the WT1 peptide, particularly the WT1_(RMF) peptide shown inSEQ ID NO: 78.

In one embodiment, said epitope comprises at least three amino acidresidues of the WT1 peptide, wherein said at least three amino acidresidues are selected from the amino acid residues corresponding toamino acid residues R1, M2, P4, N5, A6 and Y8 of the WT1_(RMF) peptideshown in SEQ ID NO: 78.

In one embodiment, said epitope comprises at least four amino acidresidues of the WT1 peptide, wherein said at least four amino acidresidues are selected from the amino acid residues corresponding toamino acid residues R1, M2, P4, N5, A6 and Y8 of the WT1_(R1) peptideshown in SEQ ID NO: 78.

In one embodiment, said epitope comprises at least five amino acidresidues of the WT1 peptide, wherein said at least five amino acidresidues are selected from the amino acid residues corresponding toamino acid residues R1, M2, P4, N5, A6 and Y8 of the WT1_(RMF) peptideshown in SEQ ID NO: 78.

In one embodiment, said epitope comprises at least six amino acidresidues of the WT1 peptide, wherein said at least six amino acidresidues are selected from the amino acid residues corresponding toamino acid residues R1, M2, P4, N5, A6 and Y8 of the WT1_(R1) peptideshown in SEQ ID NO: 78.

In one embodiment, said epitope comprises amino acid residuescorresponding to amino acid residues R1, N5 and A6 of the WT1_(RMF)peptide shown in SEQ ID NO: 78.

In one embodiment, said epitope comprises amino acid residuescorresponding to amino acid residues R1, M2, P4, N5, A6 and Y8 of theWT1_(RMF) peptide shown in SEQ ID NO: 78.

In one embodiment, said epitope comprises amino acid residuescorresponding to amino acid residues E58, R65, K66 and Q155 of theHLA-A2 sequence shown in SEQ ID NO: 138.

In one embodiment, said epitope comprises amino acid residuescorresponding to amino acid residues E58, R65, K66 and Q155 of theHLA-A2 sequence shown in SEQ ID NO: 138, and amino acid residuescorresponding to amino acid residues R1, N5 and A6 of the WT1_(RMF)peptide shown in SEQ ID NO: 78.

In one embodiment, said epitope comprises amino acid residuescorresponding to amino acid residues E58, R65, K66, Q155 and D61 of theHLA-A2 sequence shown in SEQ ID NO: 138. In one embodiment, said epitopecomprises amino acid residues corresponding to amino acid residues E58,R65, K66, Q155 and A69 of the HLA-A2 sequence shown in SEQ ID NO: 138.In one embodiment, said epitope comprises amino acid residuescorresponding to amino acid residues E58, R65, K66, Q155 and A150 of theHLA-A2 sequence shown in SEQ ID NO: 138.

In one embodiment, said epitope comprises amino acid residuescorresponding to amino acid residues E58, R65, K66, Q155, and one ormore of D61, A69 and A150 of the HLA-A2 sequence shown in SEQ ID NO:138, and amino acid residues corresponding to amino acid residues R1,M2, P4, N5, A6 and/or Y8 of the WT1_(RMF) peptide shown in SEQ ID NO:78.

In one embodiment, said epitope comprises amino acid residuescorresponding to amino acid residues E58, D61, G62, R65, K66, A69, A150,Q155, A158, T163, E166, W167 and R170 of the HLA-A2 sequence shown inSEQ ID NO: 138.

In one embodiment, said epitope comprises amino acid residuescorresponding to amino acid residues E58, D61, G62, R65, K66, A69, A150,Q155, A158, T163, E166, W167 and R170 of the HLA-A2 sequence shown inSEQ ID NO: 138, and amino acid residues corresponding to amino acidresidues R1, M2, P4, N5, A6 and/or Y8 of the WT1_(RMF) peptide shown inSEQ ID NO: 78.

In one embodiment, said epitope does not comprise amino acid residuescorresponding to amino acid residues G56, D106, W107, R108, E161, G162and/or R169 of the HLA-A2 sequence shown in SEQ ID NO: 138.

In a further aspect the present invention provides an antibody thatbinds to HLA-A2/WT1, wherein the antibody comprises

(i) a heavy chain variable region (VH) comprising a heavy chaincomplementary determining region (HCDR) 1 of SEQ ID NO: 1, a HCDR 2 ofSEQ ID NO: 2, and a HCDR 3 of SEQ ID NO: 3, and a light chain variableregion (VL) comprising a light chain complementarity determining region(LCDR) 1 of SEQ ID NO: 4, a LCDR 2 of SEQ ID NO: 5 and a LCDR 3 of SEQID NO: 6;(ii) a VH comprising a HCDR 1 of SEQ ID NO: 9, a HCDR 2 of SEQ ID NO:10, and a HCDR 3 of SEQ ID NO: 11, and a VL comprising a LCDR 1 of SEQID NO: 12, a LCDR 2 of SEQ ID NO: 13 and a LCDR 3 of SEQ ID NO: 14;(iii) a VH comprising a HCDR 1 of SEQ ID NO: 17, a HCDR 2 of SEQ ID NO:18, and a HCDR 3 of SEQ ID NO: 19, and a VL comprising a LCDR 1 of SEQID NO: 20, a LCDR 2 of SEQ ID NO: 21 and a LCDR 3 of SEQ ID NO: 22;(iv) a VH comprising a HCDR 1 of SEQ ID NO: 25, a HCDR 2 of SEQ ID NO:26, and a HCDR 3 of SEQ ID NO: 27, and a VL comprising a LCDR 1 of SEQID NO: 28, a LCDR 2 of SEQ ID NO: 29 and a LCDR 3 of SEQ ID NO: 30;(v) a VH comprising a HCDR 1 of SEQ ID NO: 33, a HCDR 2 of SEQ ID NO:34, and a HCDR 3 of SEQ ID NO: 35, and a VL comprising a LCDR 1 of SEQID NO: 36, a LCDR 2 of SEQ ID NO: 37 and a LCDR 3 of SEQ ID NO: 38;(vi) a VH comprising a HCDR 1 of SEQ ID NO: 41, a HCDR 2 of SEQ ID NO:42, and a HCDR 3 of SEQ ID NO: 43, and a VL comprising a LCDR 1 of SEQID NO: 44, a LCDR 2 of SEQ ID NO: 45 and a LCDR 3 of SEQ ID NO: 46;(vii) a VH comprising a HCDR 1 of SEQ ID NO: 49, a HCDR 2 of SEQ ID NO:50, and a HCDR 3 of SEQ ID NO: 51, and a VL comprising a LCDR 1 of SEQID NO: 52, a LCDR 2 of SEQ ID NO: 53 and a LCDR 3 of SEQ ID NO: 54;(viii) a VH comprising a HCDR 1 of SEQ ID NO: 57, a HCDR 2 of SEQ ID NO:58, and a HCDR 3 of SEQ ID NO: 59, and a VL comprising a LCDR 1 of SEQID NO: 60, a LCDR 2 of SEQ ID NO: 61 and a LCDR 3 of SEQ ID NO: 62; or(ix) a VH comprising a HCDR 1 of SEQ ID NO: 65, a HCDR 2 of SEQ ID NO:66, and a HCDR 3 of SEQ ID NO: 67, and a VL comprising a LCDR 1 of SEQID NO: 68, a LCDR 2 of SEQ ID NO: 69 and a LCDR 3 of SEQ ID NO: 70.

In a particular embodiment, the antibody comprises a VH comprising aHCDR 1 of SEQ ID NO: 1, a HCDR 2 of SEQ ID NO: 2, and a HCDR 3 of SEQ IDNO: 3, and a VL comprising a LCDR 1 of SEQ ID NO: 4, a LCDR 2 of SEQ IDNO: 5 and a LCDR 3 of SEQ ID NO: 6.

In another embodiment, the antibody comprises a VH comprising a HCDR 1of SEQ ID NO: 9, a HCDR 2 of SEQ ID NO: 10, and a HCDR 3 of SEQ ID NO:11, and a VL comprising a LCDR 1 of SEQ ID NO: 12, a LCDR 2 of SEQ IDNO: 13 and a LCDR 3 of SEQ ID NO: 14.

In a further embodiment, the antibody comprises a VH comprising a HCDR 1of SEQ ID NO: 17, a HCDR 2 of SEQ ID NO: 18, and a HCDR 3 of SEQ ID NO:19, and a VL comprising a LCDR 1 of SEQ ID NO: 20, a LCDR 2 of SEQ IDNO: 21 and a LCDR 3 of SEQ ID NO: 22.

In still a further embodiment, the antibody comprises a VH comprising aHCDR 1 of SEQ ID NO: 25, a HCDR 2 of SEQ ID NO: 26, and a HCDR 3 of SEQID NO: 27, and a VL comprising a LCDR 1 of SEQ ID NO: 28, a LCDR 2 ofSEQ ID NO: 29 and a LCDR 3 of SEQ ID NO: 30.

In some embodiments, the antibody is a human antibody. In oneembodiment, the VH is a human VH and/or the VL is a human VL. In oneembodiment, the antibody comprises CDRs as in any of the aboveembodiments, and further comprises a human framework, e.g. a humanimmunoglobulin framework.

In one embodiment, the antibody comprises

(i) a VH comprising an amino acid sequence that is at least about 95%,96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQID NO: 7, and a VL comprising an amino acid sequence that is at leastabout 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acidsequence of SEQ ID NO: 8;(ii) a VH comprising an amino acid sequence that is at least about 95%,96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQID NO: 15, and a VL comprising an amino acid sequence that is at leastabout 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acidsequence of SEQ ID NO: 16;(iii) a VH comprising an amino acid sequence that is at least about 95%,96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQID NO: 23, and a VL comprising an amino acid sequence that is at leastabout 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acidsequence of SEQ ID NO: 24;(iv) a VH comprising an amino acid sequence that is at least about 95%,96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQID NO: 31, and a VL comprising an amino acid sequence that is at leastabout 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acidsequence of SEQ ID NO: 32;(v) a VH comprising an amino acid sequence that is at least about 95%,96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQID NO: 39, and a VL comprising an amino acid sequence that is at leastabout 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acidsequence of SEQ ID NO: 40;(vi) a VH comprising an amino acid sequence that is at least about 95%,96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQID NO: 47, and a VL comprising an amino acid sequence that is at leastabout 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acidsequence of SEQ ID NO: 48;(vii) a VH comprising an amino acid sequence that is at least about 95%,96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQID NO: 55, and a VL comprising an amino acid sequence that is at leastabout 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acidsequence of SEQ ID NO: 56;(viii) a VH comprising an amino acid sequence that is at least about95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence ofSEQ ID NO: 63, and a VL comprising an amino acid sequence that is atleast about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acidsequence of SEQ ID NO: 64; or(ix) a VH comprising an amino acid sequence that is at least about 95%,96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQID NO: 71, and a VL comprising an amino acid sequence that is at leastabout 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acidsequence of SEQ ID NO: 72.

In a particular embodiment, the antibody comprises a VH comprising anamino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or100% identical to the amino acid sequence of SEQ ID NO: 7, and a VLcomprising an amino acid sequence that is at least about 95%, 96%, 97%,98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 8.

In another embodiment, the antibody comprises a VH comprising an aminoacid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO: 15, and a VLcomprising an amino acid sequence that is at least about 95%, 96%, 97%,98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 16.

In a further embodiment, the antibody comprises a VH comprising an aminoacid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO: 23, and a VLcomprising an amino acid sequence that is at least about 95%, 96%, 97%,98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 24.

In still a further embodiment, the antibody comprises a VH comprising anamino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or100% identical to the amino acid sequence of SEQ ID NO: 31, and a VLcomprising an amino acid sequence that is at least about 95%, 96%, 97%,98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 32.

In one embodiment, the antibody comprises

(i) a VH sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO: 7, and a VL sequencethat is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to theamino acid sequence of SEQ ID NO: 8;(ii) a VH sequence that is at least about 95%, 96%, 97%, 98%, 99% or100% identical to the amino acid sequence of SEQ ID NO: 15, and a VLsequence that is at least about 95%, 96%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO: 16;(iii) a VH sequence that is at least about 95%, 96%, 97%, 98%, 99% or100% identical to the amino acid sequence of SEQ ID NO: 23, and a VLsequence that is at least about 95%, 96%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO: 24;(iv) a VH sequence that is at least about 95%, 96%, 97%, 98%, 99% or100% identical to the amino acid sequence of SEQ ID NO: 31, and a VLsequence that is at least about 95%, 96%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO: 32;(v) a VH sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO: 39, and a VL sequencethat is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to theamino acid sequence of SEQ ID NO: 40;(vi) a VH sequence that is at least about 95%, 96%, 97%, 98%, 99% or100% identical to the amino acid sequence of SEQ ID NO: 47, and a VLsequence that is at least about 95%, 96%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO: 48;(vii) a VH sequence that is at least about 95%, 96%, 97%, 98%, 99% or100% identical to the amino acid sequence of SEQ ID NO: 55, and a VLsequence that is at least about 95%, 96%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO: 56;(viii) a VH sequence that is at least about 95%, 96%, 97%, 98%, 99% or100% identical to the amino acid sequence of SEQ ID NO: 63, and a VLsequence that is at least about 95%, 96%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO: 64; or(ix) a VH sequence that is at least about 95%, 96%, 97%, 98%, 99% or100% identical to the amino acid sequence of SEQ ID NO: 71, and a VLsequence that is at least about 95%, 96%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO: 72.

In a particular embodiment, the antibody comprises a VH sequence that isat least about 95%, 96%, 97%, 98%, 99% or 100% identical to the aminoacid sequence of SEQ ID NO: 7, and a VL sequence that is at least about95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence ofSEQ ID NO: 8.

In another embodiment, the antibody comprises a VH sequence that is atleast about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acidsequence of SEQ ID NO: 15, and a VL sequence that is at least about 95%,96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQID NO: 16.

In a further embodiment, the antibody comprises a VH sequence that is atleast about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acidsequence of SEQ ID NO: 23, and a VL sequence that is at least about 95%,96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQID NO: 24.

In still a further embodiment, the antibody comprises a VH sequence thatis at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the aminoacid sequence of SEQ ID NO: 31, and a VL sequence that is at least about95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence ofSEQ ID NO: 32.

In certain embodiments, a VH or VL sequence having at least 95%, 96%,97%, 98%, or 99% identity contains substitutions (e.g., conservativesubstitutions), insertions, or deletions relative to the referencesequence, but an antibody comprising that sequence retains the abilityto bind to HLA-A2/WT1. In certain embodiments, a total of 1 to 10 aminoacids have been substituted, inserted and/or deleted in the VH (SEQ IDNO: 7, 15, 23, 31, 39, 47, 55, 63 or 71) and/or a total of 1 to 10 aminoacids have been substituted, inserted and/or deleted in the VL (SEQ IDNO: 8, 16, 24, 32, 40, 48, 56, 64 or 72). In certain embodiments,substitutions, insertions, or deletions occur in regions outside theHVRs (i.e., in the FRs). Optionally, the antibody comprises the VHsequence and/or the VL sequence indicated above, includingpost-translational modifications of that sequence.

In one embodiment, the antibody comprises

(i) a VH comprising the amino acid sequence of SEQ ID NO: 7, and a VLcomprising the amino acid sequence of SEQ ID NO: 8;(ii) a VH comprising the amino acid sequence of SEQ ID NO: 15, and a VLcomprising the amino acid sequence of SEQ ID NO: 16;(iii) a VH comprising the amino acid sequence of SEQ ID NO: 23, and a VLcomprising the amino acid sequence of SEQ ID NO: 24;(iv) a VH comprising the amino acid sequence of SEQ ID NO: 31, and a VLcomprising the amino acid sequence of SEQ ID NO: 32;(v) a VH comprising the amino acid sequence of SEQ ID NO: 39, and a VLcomprising the amino acid sequence of SEQ ID NO: 40;(vi) a VH comprising the amino acid sequence of SEQ ID NO: 47, and a VLcomprising the amino acid sequence of SEQ ID NO: 48;(vii) a VH comprising the amino acid sequence of SEQ ID NO: 55, and a VLcomprising the amino acid sequence of SEQ ID NO: 56;(viii) a VH comprising the amino acid sequence of SEQ ID NO: 63, and aVL comprising the amino acid sequence of SEQ ID NO: 64; or(ix) a VH comprising the amino acid sequence of SEQ ID NO: 71, and a VLcomprising the amino acid sequence of SEQ ID NO: 72.

In one embodiment, the antibody comprises

(i) the VH sequence of SEQ ID NO: 7, and the VL sequence of SEQ ID NO:8;(ii) the VH sequence of SEQ ID NO: 15, and the VL sequence of SEQ ID NO:16;(iii) the VH sequence of SEQ ID NO: 23, and the VL sequence of SEQ IDNO: 24;(iv) the VH sequence of SEQ ID NO: 31, and the VL sequence of SEQ ID NO:32;(v) the VH sequence of SEQ ID NO: 39, and the VL sequence of SEQ ID NO:40;(vi) the VH sequence of SEQ ID NO: 47, and the VL sequence of SEQ ID NO:48;(vii) the VH sequence of SEQ ID NO: 55, and the VL sequence of SEQ IDNO: 56;(viii) the VH sequence of SEQ ID NO: 63, and the VL sequence of SEQ IDNO: 64; or(ix) the VH sequence of SEQ ID NO: 71, and the VL sequence of SEQ ID NO:72.

In a particular embodiment, the antibody comprises a VH comprising theamino acid sequence of SEQ ID NO: 7 and a VL comprising the amino acidsequence of SEQ ID NO: 8.

In another embodiment, the antibody comprises a VH comprising the aminoacid sequence of SEQ ID NO: 15 and a VL comprising the amino acidsequence of SEQ ID NO: 16.

In a further embodiment, the antibody comprises a VH comprising theamino acid sequence of SEQ ID NO: 23 and a VL comprising the amino acidsequence of SEQ ID NO: 24.

In still a further embodiment, the antibody comprises a VH comprisingthe amino acid sequence of SEQ ID NO: 31 and a VL comprising the aminoacid sequence of SEQ ID NO: 32.

In a particular embodiment, the antibody comprises the VH sequence ofSEQ ID NO: 7 and the VL sequence of SEQ ID NO: 8.

In another embodiment, the antibody comprises the VH sequence of SEQ IDNO: 15 and the VL sequence of SEQ ID NO: 16.

In a further embodiment, the antibody comprises the VH sequence of SEQID NO: 23 and the VL sequence of SEQ ID NO: 24.

In still a further embodiment, the antibody comprises the VH sequence ofSEQ ID NO: 31 and the VL sequence of SEQ ID NO: 32.

In one embodiment, the antibody comprises a human constant region. Inone embodiment, the antibody is an immunoglobulin molecule comprising ahuman constant region, particularly an IgG class immunoglobulin moleculecomprising a human CH1, CH2, CH3 and/or CL domain. Exemplary sequencesof human constant domains are given in SEQ ID NOs 112 and 113 (humankappa and lambda CL domains, respectively) and SEQ ID NO: 114 (humanIgG₁ heavy chain constant domains CH1-CH2-CH3). In some embodiments, theantibody comprises a light chain constant region comprising an aminoacid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO: 112 or SEQ ID NO:113, particularly the amino acid sequence of SEQ ID NO: 112. In someembodiments, the antibody comprises a heavy chain constant regioncomprising an amino acid sequence that is at least about 95%, 96%, 97%,98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 114.Particularly, the heavy chain constant region may comprise amino acidmutations in the Fc domain as described herein.

In one embodiment, the antibody is a monoclonal antibody.

In one embodiment, the antibody is an IgG, particularly an IgG₁,antibody. In one embodiment, the antibody is a full-length antibody.

In one embodiment, the antibody comprises an Fc domain, particularly anIgG Fc domain, more particularly an IgG1 Fc domain. In one embodimentthe Fc domain is a human Fc domain. The Fc domain of the antibody mayincorporate any of the features, singly or in combination, describedherein in relation to the Fc domain of the bispecific antigen bindingmolecule of the invention.

In another embodiment, the antibody is an antibody fragment selectedfrom the group of an Fv molecule, a scFv molecule, a Fab molecule, and aF(ab′)₂ molecule; particularly a Fab molecule. In another embodiment,the antibody fragment is a diabody, a triabody or a tetrabody.

In a further aspect, the antibody according to any of the aboveembodiments may incorporate any of the features, singly or incombination, as described in the sections below.

Glycosylation Variants

In certain embodiments, an antibody provided herein is altered toincrease or decrease the extent to which the antibody is glycosylated.Addition or deletion of glycosylation sites to an antibody may beconveniently accomplished by altering the amino acid sequence such thatone or more glycosylation sites is created or removed.

Where the antibody comprises an Fc region, the oligosaccharide attachedthereto may be altered. Native antibodies produced by mammalian cellstypically comprise a branched, biantennary oligosaccharide that isgenerally attached by an N-linkage to Asn297 of the CH2 domain of the Fcregion. See, e.g., Wright et al. TIBTECH 15:26-32 (1997). Theoligosaccharide may include various carbohydrates, e.g., mannose,N-acetyl glucosamine (GlcNAc), galactose, and sialic acid, as well as afucose attached to a GlcNAc in the “stem” of the biantennaryoligosaccharide structure. In some embodiments, modifications of theoligosaccharide in an antibody of the invention may be made in order tocreate antibody variants with certain improved properties.

In one embodiment, antibody variants are provided having anon-fucosylated oligosaccharide, i.e. an oligosaccharide structure thatlacks fucose attached (directly or indirectly) to an Fc region. Suchnon-fucosylated oligosaccharide (also referred to as “afucosylated”oligosaccharide) particularly is an N-linked oligosaccharide which lacksa fucose residue attached to the first GlcNAc in the stem of thebiantennary oligosaccharide structure. In one embodiment, antibodyvariants are provided having an increased proportion of non-fucosylatedoligosaccharides in the Fc region as compared to a native or parentantibody. For example, the proportion of non-fucosylatedoligosaccharides may be at least about 20%, at least about 40%, at leastabout 60%, at least about 80%, or even about 100% (i.e. no fucosylatedoligosaccharides are present). The percentage of non-fucosylatedoligosaccharides is the (average) amount of oligosaccharides lackingfucose residues, relative to the sum of all oligosaccharides attached toAsn 297 (e. g. complex, hybrid and high mannose structures) as measuredby MALDI-TOF mass spectrometry, as described in WO 2006/082515, forexample. Asn297 refers to the asparagine residue located at aboutposition 297 in the Fc region (EU numbering of Fc region residues);however, Asn297 may also be located about ±3 amino acids upstream ordownstream of position 297, i.e., between positions 294 and 300, due tominor sequence variations in antibodies. Such antibodies having anincreased proportion of non-fucosylated oligosaccharides in the Fcregion may have improved FcγRIIIa receptor binding and/or improvedeffector function, in particular improved ADCC function. See, e.g., US2003/0157108; US 2004/0093621.

Examples of cell lines capable of producing antibodies with reducedfucosylation include Lec13 CHO cells deficient in protein fucosylation(Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986); US2003/0157108; and WO 2004/056312, especially at Example 11), andknockout cell lines, such as alpha-1,6-fucosyltransferase gene, FUT8,knockout CHO cells (see, e.g., Yamane-Ohnuki et al. Biotech. Bioeng.87:614-622 (2004); Kanda, Y. et al., Biotechnol. Bioeng., 94(4):680-688(2006); and WO2003/085107), or cells with reduced or abolished activityof a GDP-fucose synthesis or transporter protein (see, e.g.,US2004259150, US2005031613, US2004132140, US2004110282).

In a further embodiment, antibody variants are provided with bisectedoligosaccharides, e.g., in which a biantennary oligosaccharide attachedto the Fc region of the antibody is bisected by GlcNAc. Such antibodyvariants may have reduced fucosylation and/or improved ADCC function asdescribed above. Examples of such antibody variants are described, e.g.,in Umana et al., Nat Biotechnol 17, 176-180 (1999); Ferrara et al.,Biotechn Bioeng 93, 851-861 (2006); WO 99/54342; WO 2004/065540, WO2003/011878.

Antibody variants with at least one galactose residue in theoligosaccharide attached to the Fc region are also provided. Suchantibody variants may have improved CDC function. Such antibody variantsare described, e.g., in WO 1997/30087; WO 1998/58964; and WO 1999/22764.

Cysteine Engineered Antibody Variants

In certain embodiments, it may be desirable to create cysteineengineered antibodies, e.g., “thioMAbs,” in which one or more residuesof an antibody are substituted with cysteine residues. In particularembodiments, the substituted residues occur at accessible sites of theantibody. By substituting those residues with cysteine, reactive thiolgroups are thereby positioned at accessible sites of the antibody andmay be used to conjugate the antibody to other moieties, such as drugmoieties or linker-drug moieties, to create an immunoconjugate, asdescribed further herein. Cysteine engineered antibodies may begenerated as described, e.g., in U.S. Pat. Nos. 7,521,541, 8,30,930,7,855,275, 9,000,130, or WO2016040856.

Antibody Derivatives

In certain embodiments, an antibody provided herein may be furthermodified to contain additional nonproteinaceous moieties that are knownin the art and readily available. The moieties suitable forderivatization of the antibody include but are not limited to watersoluble polymers. Non-limiting examples of water soluble polymersinclude, but are not limited to, polyethylene glycol (PEG), copolymersof ethylene glycol/propylene glycol, carboxymethylcellulose, dextran,polyvinyl alcohol, polyvinyl pyrrolidone, poly-1, 3-dioxolane,poly-1,3,6-trioxane, ethylene/maleic anhydride copolymer, polyaminoacids(either homopolymers or random copolymers), and dextran or poly(n-vinylpyrrolidone)polyethylene glycol, propropylene glycol homopolymers,prolypropylene oxide/ethylene oxide co-polymers, polyoxyethylatedpolyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof.Polyethylene glycol propionaldehyde may have advantages in manufacturingdue to its stability in water. The polymer may be of any molecularweight, and may be branched or unbranched. The number of polymersattached to the antibody may vary, and if more than one polymer areattached, they can be the same or different molecules. In general, thenumber and/or type of polymers used for derivatization can be determinedbased on considerations including, but not limited to, the particularproperties or functions of the antibody to be improved, whether theantibody derivative will be used in a therapy under defined conditions,etc.

In another embodiment, conjugates of an antibody and nonproteinaceousmoiety that may be selectively heated by exposure to radiation areprovided. In one embodiment, the nonproteinaceous moiety is a carbonnanotube (Kam et al., Proc. Natl. Acad. Sci. USA 102: 11600-11605(2005)). The radiation may be of any wavelength, and includes, but isnot limited to, wavelengths that do not harm ordinary cells, but whichheat the nonproteinaceous moiety to a temperature at which cellsproximal to the antibody-nonproteinaceous moiety are killed.

Immunoconjugates

The invention also provides immunoconjugates comprising ananti-HLA-A2/WT1 antibody as described herein conjugated (chemicallybonded) to one or more therapeutic agents such as cytotoxic agents,chemotherapeutic agents, drugs, growth inhibitory agents, toxins (e.g.,protein toxins, enzymatically active toxins of bacterial, fungal, plant,or animal origin, or fragments thereof), or radioactive isotopes.

In one embodiment, an immunoconjugate is an antibody-drug conjugate(ADC) in which an antibody is conjugated to one or more of thetherapeutic agents mentioned above. The antibody is typically connectedto one or more of the therapeutic agents using linkers. An overview ofADC technology including examples of therapeutic agents and drugs andlinkers is set forth in Pharmacol Review 68:3-19 (2016).

In another embodiment, an immunoconjugate comprises an antibody asdescribed herein conjugated to an enzymatically active toxin or fragmentthereof, including but not limited to diphtheria A chain, nonbindingactive fragments of diphtheria toxin, exotoxin A chain (from Pseudomonasaeruginosa), ricin A chain, abrin A chain, modeccin A chain,alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolacaamericana proteins (PAPI, PAPII, and PAP-S), momordica charantiainhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin,mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes.

In another embodiment, an immunoconjugate comprises an antibody asdescribed herein conjugated to a radioactive atom to form aradioconjugate. A variety of radioactive isotopes are available for theproduction of radioconjugates. Examples include At²¹¹, I¹³¹, I¹²⁵, Y⁹⁰,Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³, Bi²¹², P³², Pb²¹² and radioactive isotopes of Lu.When the radioconjugate is used for detection, it may comprise aradioactive atom for scintigraphic studies, for example tc99m or I123,or a spin label for nuclear magnetic resonance (NMR) imaging (also knownas magnetic resonance imaging, mri), such as iodine-123 again,iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17,gadolinium, manganese or iron.

Conjugates of an antibody and cytotoxic agent may be made using avariety of bifunctional protein coupling agents such asN-succinimidyl-3-(2-pyridyldithio) propionate (SPDP),succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC),iminothiolane (IT), bifunctional derivatives of imidoesters (such asdimethyl adipimidate HCl), active esters (such as disuccinimidylsuberate), aldehydes (such as glutaraldehyde), bis-azido compounds (suchas bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (suchas bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astoluene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin canbe prepared as described in Vitetta et al., Science 238:1098 (1987).Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent forconjugation of radionucleotide to the antibody. See WO94/11026. Thelinker may be a “cleavable linker” facilitating release of a cytotoxicdrug in the cell. For example, an acid-labile linker,peptidase-sensitive linker, photolabile linker, dimethyl linker ordisulfide-containing linker (Chari et al., Cancer Res. 52:127-131(1992); U.S. Pat. No. 5,208,020) may be used.

The immunuoconjugates or ADCs herein expressly contemplate, but are notlimited to such conjugates prepared with cross-linker reagentsincluding, but not limited to, BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS,MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS,sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, and sulfo-SMPB, and SVSB(succinimidyl-(4-vinylsulfone)benzoate) which are commercially available(e.g., from Pierce Biotechnology, Inc., Rockford, Ill., U.S.A).

Multispecific Antibodies

In certain embodiments, an antibody provided herein is a multispecificantibody, e.g. a bispecific antibody. Multispecific antibodies aremonoclonal antibodies that have binding specificities for at least twodifferent sites, i.e., different epitopes on different antigens ordifferent epitopes on the same antigen. In certain embodiments, themultispecific antibody has three or more binding specificities. Incertain embodiments, one of the binding specificities is for HLA-A2/WT1and the other (two or more) specificity is for any other antigen. Incertain embodiments, bispecific antibodies may bind to two (or more)different epitopes of HLA-A2/WT1. Multispecific (e.g., bispecific)antibodies may also be used to localize cytotoxic agents or cells tocells which express HLA-A2/WT1. Multispecific antibodies can be preparedas full length antibodies or antibody fragments.

Techniques for making multispecific antibodies include, but are notlimited to, recombinant co-expression of two immunoglobulin heavychain-light chain pairs having different specificities (see Milstein andCuello, Nature 305: 537 (1983)) and “knob-in-hole” engineering (see,e.g., U.S. Pat. No. 5,731,168, and Atwell et al., J. Mol. Biol. 270:26(1997)). Multi-specific antibodies may also be made by engineeringelectrostatic steering effects for making antibody Fc-heterodimericmolecules (see, e.g., WO 2009/089004); cross-linking two or moreantibodies or fragments (see, e.g., U.S. Pat. No. 4,676,980, and Brennanet al., Science, 229: 81 (1985)); using leucine zippers to producebi-specific antibodies (see, e.g., Kostelny et al., J. Immunol.,148(5):1547-1553 (1992) and WO 2011/034605); using the common lightchain technology for circumventing the light chain mis-pairing problem(see, e.g., WO 98/50431); using “diabody” technology for makingbispecific antibody fragments (see, e.g., Hollinger et al., Proc. Natl.Acad. Sci. USA, 90:6444-6448 (1993)); and using single-chain Fv (sFv)dimers (see, e.g. Gruber et al., J. Immunol., 152:5368 (1994)); andpreparing trispecific antibodies as described, e.g., in Tutt et al. J.Immunol. 147: 60 (1991).

Engineered antibodies with three or more antigen binding sites,including for example, “Octopus antibodies,” or DVD-Ig are also includedherein (see, e.g. WO 2001/77342 and WO 2008/024715). Other examples ofmultispecific antibodies with three or more antigen binding sites can befound in WO 2010/115589, WO 2010/112193, WO 2010/136172, WO2010/145792,and WO 2013/026831. The bispecific antibody or antigen binding fragmentthereof also includes a “Dual Acting FAb” or “DAF” comprising an antigenbinding site that binds to HLA-A2/WT1 as well as another differentantigen, or two different epitopes of HLA-A2/WT1 (see, e.g., US2008/0069820 and WO 2015/095539).

Multi-specific antibodies may also be provided in an asymmetric formwith a domain crossover in one or more binding arms of the same antigenspecificity, i.e. by exchanging the VH/VL domains (see e.g., WO2009/080252 and WO 2015/150447), the CH1/CL domains (see e.g., WO2009/080253) or the complete Fab arms (see e.g., WO 2009/080251, WO2016/016299, also see Schaefer et al, PNAS, 108 (2011) 1187-1191, andKlein at al., MAbs 8 (2016) 1010-20). Asymmetrical Fab arms can also beengineered by introducing charged or non-charged amino acid mutationsinto domain interfaces to direct correct Fab pairing. See e.g., WO2016/172485.

Various further molecular formats for multispecific antibodies are knownin the art and are included herein (see e.g., Spiess et al., Mol Immunol67 (2015) 95-106).

A particular type of multispecific antibodies, also included herein, arebispecific antibodies designed to simultaneously bind to a surfaceantigen on a target cell, e.g., a tumor cell, and to an activating,invariant component of the T cell receptor (TCR) complex, such as CD3,for retargeting of T cells to kill target cells. Hence, in certainembodiments, an antibody provided herein is a multispecific antibody,particularly a bispecific antibody, wherein one of the bindingspecificities is for HLA-A2/WT1 and the other is for CD3.

Examples of bispecific antibody formats that may be useful for thispurpose include, but are not limited to, the so-called “BiTE”(bispecific T cell engager) molecules wherein two scFv molecules arefused by a flexible linker (see, e.g., WO2004/106381, WO2005/061547,WO2007/042261, and WO2008/119567, Nagorsen and Bäuerle, Exp Cell Res317, 1255-1260 (2011)); diabodies (Holliger et al., Prot Eng 9, 299-305(1996)) and derivatives thereof, such as tandem diabodies (“TandAb”;Kipriyanov et al., J Mol Biol 293, 41-56 (1999)); “DART” (dual affinityretargeting) molecules which are based on the diabody format but featurea C-terminal disulfide bridge for additional stabilization (Johnson etal., J Mol Biol 399, 436-449 (2010)), and so-called triomabs, which arewhole hybrid mouse/rat IgG molecules (reviewed in Seimetz et al., CancerTreat Rev 36, 458-467 (2010)). Particular T cell bispecific antibodyformats included herein are described in WO 2013/026833, WO2013/026839,WO 2016/020309; Bacac et al., Oncoimmunology 5(8) (2016) e1203498.

Bispecific Antigen Binding Molecules that Bind to HLA-A2/WT1 and aSecond Antigen

The invention also provides a bispecific antigen binding molecule, i.e.an antigen binding molecule that comprises at least two antigen bindingmoieties capable of specific binding to two distinct antigenicdeterminants (a first and a second antigen).

Based on the HLA-A2/WT1 antibodies they developed, the present inventorshave developed bispecific antigen binding molecules that bind toHLA-A2/WT1 and a further antigen, particularly an activating T cellantigen such as CD3.

As shown in the Examples, these bispecific antigen binding moleculeshave a number of remarkable properties, including good efficacy and lowtoxicity.

Thus, in certain aspects, the invention provides a bispecific antigenbinding molecule, comprising (a) a first antigen binding moiety thatbinds to a first antigen, wherein the first antigen is HLA-A2/WT1, and(b) a second antigen binding moiety which specifically binds to a secondantigen, wherein the bispecific antigen binding molecule has any of thefollowing features.

The bispecific antigen binding molecule of the invention specificallyinduces T-cell mediated killing of cells expressing HLA-A2/WT1 (i.e. acomplex of a HLA-A2 molecule and a WT1-derived peptide). In someembodiments, the bispecific antigen binding molecule of the inventionspecifically induces T-cell mediated killing of cells expressingHLA-A2/WT1_(RMF) (i.e. a complex of a HLA-A2 molecule and the WT1_(RMF)peptide). In a more specific embodiment, the bispecific antigen bindingmolecule specifically induces T-cell mediated killing of cellsexpressing HLA-A2.01/WT1_(RMF) (i.e. a complex of a HLA-A201 moleculeand the WT1_(RMF) peptide).

In one embodiment, induction of T-cell mediated killing by thebispecific antigen binding molecule is determined using HLA-A2/peptide(e.g. WT1_(RMF) or WT1_(VLD) peptide)-expressing cells, particularlypeptide-pulsed T2 cells, and measuring lactate dehydrogenase (LDH)release from said cells after incubation with the bispecific antigenbinding molecule in the presence of T cells.

In a specific embodiment, induction of T-cell mediated killing by thebispecific antigen binding molecule is determined as follows:

T2 cells (ATCC, Cat. No. CRL-1992) are prepared as a cell suspension at10⁶ cells/ml in IMDM medium (Gibco by Life Technologies, Cat No.31980-048), supplemented with 10% FBS (Gibco, Cat No. 16140-071)+1%Penicillin-Streptomycin (Gibco, Cat No. 15070-063) (complete medium).Cells are kept in a total volume of 10 ml in a tube, and incubated with10 μl of peptide (e.g. WT1_(VLD) peptide (SEQ ID NO: 77), or RMF peptide(SEQ ID NO: 78)) at 10⁻² M (final concentration of the peptide: 10⁻⁵M)for 2 hours at 37° C. with 5% CO₂. Pan CD3⁺ cells are purified fromPBMCs isolated from buffy coat by Ficoll (GE Healthcare, Cat. No.17-1440-03) gradient centrifugation. Total CD3⁺ T cells are purified byMACS (Miltenyi Biotec) using a Human Pan T cell Isolation Kit (MiltenyiBiotec, Cat. No. 130-096-535). The cytotoxicity assay is performed asfollows: The peptide-pulsed cells (100 μl) are seeded into a 96 wellmicrotiter round bottom plate (3×10⁵ cells/ml), co-cultured with 50 μlof T cells (6×10⁶ cells/ml), and with 50 μl of titrated bispecificantigen binding molecule (e.g. at 40 μg/ml to 0.00004 μg/ml) in completemedium for 18 hours at 37° C. with 5% CO₂. Thereafter, 50 μl ofsupernatant are transferred into a new white plate, and 25 μl per wellof CytoTox-Glo Luciferase Assay (Promega, Cat. No. G9291) are added forincubation at room temperature (RT) for 15 minutes. The luminescencesignal (for measurement of LDH release as indicative of cell death) isread by EnVision (PerkinElmer). Data are presented as RelativeLuminescence Unit (RLU).

The bispecific antigen binding molecule of the invention specificallyactivates T cells in the presence of cells expressing HLA-A2/WT1 (i.e. acomplex of a HLA-A2 molecule and a WT1-derived peptide). In someembodiments, the bispecific antigen binding molecule of the inventionspecifically activates T cells in the presence of cells expressingHLA-A2/WT1_(RMF) (i.e. a complex of a HLA-A2 molecule and the WT1_(RMF)peptide). In a more specific embodiment, the bispecific antigen bindingmolecule specifically activates T cells in the presence of cellsexpressing HLA-A201/WT1_(RMF) (i.e. a complex of a HLA-A201 molecule andthe WT1_(RMF) peptide).

In one embodiment, activation of T cells by the bispecific antigenbinding molecule is determined by measuring, particularly by flowcytometry, expression of CD25 and/or CD69 by T cells after incubationwith the bispecific antigen binding molecule in the presence ofHLA-A2/peptide (e.g. WT1_(RMF) or WT1_(VLD) peptide)-expressing cells,particularly peptide-pulsed T2 cells.

In a specific embodiment, activation of T cells by the bispecificantigen binding molecule is determined as follows:

T2 cells (ATCC, Cat. No. CRL-1992) are prepared as a cell suspension at10⁶ cells/ml in IMDM medium (Gibco by Life Technologies, Cat No.31980-048), supplemented with 10% FBS (Gibco, Cat No. 16140-071)+1%Penicillin-Streptomycin (Gibco, Cat No. 15070-063) (complete medium).Cells are kept in a total volume of 10 ml in a tube, and incubated with10 μl of peptide (e.g. WT1_(VLD) peptide (SEQ ID NO: 77), or RMF peptide(SEQ ID NO: 78)) at 10⁻² M (final concentration of the peptide: 10⁻⁵M)for 2 hours at 37° C. with 5% CO₂. Pan CD3⁺ cells are purified fromPBMCs isolated from buffy coat by Ficoll (GE Healthcare, Cat. No.17-1440-03) gradient centrifugation. Total CD3⁺ T cells are purified byMACS (Miltenyi Biotec) using a Human Pan T cell Isolation Kit (MiltenyiBiotec, Cat. No. 130-096-535). The cytotoxicity assay is performed asfollows: The peptide-pulsed cells (100 μl) are seeded into a 96 wellmicrotiter round bottom plate (3×10⁵ cells/ml), co-cultured with 50 μlof T cells (6×10⁶ cells/ml), and with 50 μl of titrated bispecificantigen binding molecule (e.g. at 40 μg/ml to 0.00004 μg/ml) in completemedium for 18 hours at 37° C. with 5% CO₂. Cells are harvested after 18hours of co-incubation, and stained with antibodies against CD3(Biolegend Cat. No. 300321), CD25 (Biolegend Cat. No. 302606) and CD69(Biolegend Cat. No. 310914) to measure T cell activation by flowcytometry.

In another embodiment, activation of T cells by the bispecific antigenbinding molecule is determined using HLA-A2/peptide (e.g. WT1_(RMF) orWT1_(VLD) peptide)-expressing cells, particularly peptide-pulsed T2cells, and a reporter T cell line, particularly a Jurkat T cell linethat expresses a luciferase reporter driven by an NFAT (nuclear factorof activated T cells) response element.

In a specific embodiment, activation of T cells by the bispecificantigen binding molecule is determined as follows:

T2 cells (ATCC, Cat. No. CRL-1992) are prepared as a cell suspension at10⁶ cells/ml in IMDM medium (Gibco by Life Technologies, Cat No.31980-048), supplemented with 10% FBS (Gibco, Cat No. 16140-071)+1%Penicillin-Streptomycin (Gibco, Cat No. 15070-063) (complete medium).Cells are kept in a total volume of 10 ml in a tube, and incubated with10 μl of peptide (e.g. WT1_(VLD) peptide (SEQ ID NO: 77), or RMF peptide(SEQ ID NO: 78)) at 10⁻² M (final concentration of the peptide: 10⁻⁵M)for 2 hours at 37° C. with 5% CO₂. After washing, 90 μl of thepeptide-pulsed cells in a cell suspension of 2.2×10⁵ cells/ml are seededinto a 96 well microtiter round bottom plate (20,000 cells/well, TPP,Cat. No. 92097), co-cultured with 50 μl of Jurkat cells that expressluciferase under the promoter of NFAT (Jurkat-NFAT; Promega, Cat. No.CS176501) (cell suspension of 2×10⁶ cells/ml), and with 10 μl oftitrated bispecific antigen binding molecule (e.g. at 100 μg/ml to0.0064 μg/ml in PBS) for 16 hours at 37° C. with 5% CO₂. Thereafter, 50μl of supernatant are removed, and replaced with 100 μl per well ofBright-Glo Luciferase Assay (Promega, Cat. No. E2620) for incubation atroom temperature (RT). Five minutes later, 180 μl of supernatant aretransferred into a new white plate to measure luminescence signal byEnVision (PerkinElmer). Data are presented as Relative Luminescence Unit(RLU).

In one embodiment, the bispecific antigen binding molecule of theinvention does not significantly induce T cell mediated killing of, oractivate T cells in the presence of, cells expressing HLA-A2 alone (i.e.without a peptide) or HLA-A2 with a peptide other than a WT1-derivedpeptide such as WT1_(RMF) or WT1_(VLD).

In one embodiment, the bispecific antigen binding does not significantlyinduce T cell mediated killing of, or activate T cells in the presenceof, cells expressing HLA-A2 in the absence of a WT1-derived peptide,particularly WT1_(RMF) or WT1_(VLD). In one embodiment, the bispecificantigen binding molecule induces T cell mediated killing of, and/oractivates T cells in the presence of, cells expressing HLA-A2/WT1(specifically HLA-A2/WT1_(RMF) or HLA-A2/WT1_(VLD)) with an EC50 that isat least 5, at least 10, at least 15, at least 20, at least 25, at least50, at least 75 or at least 100 times lower than the EC50 for inductionof T cell mediated killing of, or activation of T cells in the presenceof, cells expressing HLA-A2 in the absence of a WT1-derived peptide(specifically WT1_(RMF) or WT1_(VLD)).

In one embodiment, the bispecific antigen binding molecule induces Tcell mediated killing of, and/or activates T cells in the presence of,cells expressing HLA-A2/WT1 (specifically HLA-A2/WT1_(RMF) orHLA-A2/WT1_(VLD)), but does not significantly induce T cell mediatedkilling of, or activate T cells in the presence of, cells expressing toHLA-A2 with a peptide selected from the peptides in Table 5 (thepeptides of SEQ ID NOs 79-105). In one embodiment, the bispecificantigen binding molecule induces T cell mediated killing of, and/oractivates T cells in the presence of, cells expressing to HLA-A2/WT1(specifically HLA-A2/WT1_(RMF) or HLA-A2/WT1_(VLD)), but does notsignificantly induce T cell mediated killing of, or activate T cells inthe presence of, cells expressing to HLA-A2 with any of the peptides inTable 5 (the peptides of SEQ ID NOs 79-105). In one embodiment, thebispecific antigen binding molecule induces T cell mediated killing of,and/or activates T cells in the presence of, cells expressing HLA-A2/WT1(specifically HLA-A2/WT1_(RMF) or HLA-A2/WT1_(VLD)) with an EC50 that isat least 5, at least 10, at least 15, at least 20, at least 25, at least50, at least 75 or at least 100 times lower than the EC50 for inductionof T cell mediated killing of, and/or activation of T cells in thepresence of, cells expressing to HLA-A2 with a peptide selected from thepeptides in Table 5 (the peptides of SEQ ID NOs 79-105). In oneembodiment, the bispecific antigen binding molecule induces T cellmediated killing of, and/or activates T cells in the presence of, cellsexpressing HLA-A2/WT1 (specifically HLA-A2/WT1_(RMF) orHLA-A2/WT1_(VLD)) with an EC50 that is at least 5, at least 10, at least15, at least 20, at least 25, at least 50, at least 75 or at least 100times lower than the EC50 for induction of T cell mediated killing of,and/or activation of T cells in the presence of, cells expressing toHLA-A2 with any of the peptides in Table 5 (the peptides of SEQ ID NOs79-105).

In one embodiment, induction of T cell mediated killing and/oractivation of T cells is determined as described above, and the EC50 iscalculated in Microsoft® Excel using the XLfit® add-on (ID BusinessSolutions, Guildford, UK).

According to particular embodiments of the invention, the antigenbinding moieties comprised in the bispecific antigen binding moleculeare Fab molecules (i.e. antigen binding domains composed of a heavy anda light chain, each comprising a variable and a constant domain). In oneembodiment, the first and/or the second antigen binding moiety is a Fabmolecule. In one embodiment, said Fab molecule is human. In a particularembodiment, said Fab molecule is humanized. In yet another embodiment,said Fab molecule comprises human heavy and light chain constantdomains.

Preferably, at least one of the antigen binding moieties is a crossoverFab molecule. Such modification reduces mispairing of heavy and lightchains from different Fab molecules, thereby improving the yield andpurity of the bispecific antigen binding molecule of the invention inrecombinant production. In a particular crossover Fab molecule usefulfor the bispecific antigen binding molecule of the invention, thevariable domains of the Fab light chain and the Fab heavy chain (VL andVH, respectively) are exchanged. Even with this domain exchange,however, the preparation of the bispecific antigen binding molecule maycomprise certain side products due to a so-called Bence Jones-typeinteraction between mispaired heavy and light chains (see Schaefer etal, PNAS, 108 (2011) 11187-11191). To further reduce mispairing of heavyand light chains from different Fab molecules and thus increase thepurity and yield of the desired bispecific antigen binding molecule,charged amino acids with opposite charges may be introduced at specificamino acid positions in the CH1 and CL domains of either the Fabmolecule(s) binding to the first antigen (HLA-A2/WT1), or the Fabmolecule binding to the second antigen (e.g. an activating T cellantigen such as CD3), as further described herein. Charge modificationsare made either in the conventional Fab molecule(s) comprised in thebispecific antigen binding molecule (such as shown e.g. in FIGS. 1 A-C,G-J), or in the VH/VL crossover Fab molecule(s) comprised in thebispecific antigen binding molecule (such as shown e.g. in FIG. 1 D-F,K-N) (but not in both). In particular embodiments, the chargemodifications are made in the conventional Fab molecule(s) comprised inthe bispecific antigen binding molecule (which in particular embodimentsbind(s) to the first antigen, i.e. HLA-A2/WT1).

In a particular embodiment according to the invention, the bispecificantigen binding molecule is capable of simultaneous binding to the firstantigen (i.e. HLA-A2/WT1), and the second antigen (e.g. an activating Tcell antigen, particularly CD3). In one embodiment, the bispecificantigen binding molecule is capable of crosslinking a T cell and atarget cell by simultaneous binding HLA-A2/WT1 and an activating T cellantigen. In an even more particular embodiment, such simultaneousbinding results in lysis of the target cell, particularly a HLA-A2/WT1expressing tumor cell. In one embodiment, such simultaneous bindingresults in activation of the T cell. In other embodiments, suchsimultaneous binding results in a cellular response of a T lymphocyte,particularly a cytotoxic T lymphocyte, selected from the group of:proliferation, differentiation, cytokine secretion, cytotoxic effectormolecule release, cytotoxic activity, and expression of activationmarkers. In one embodiment, binding of the bispecific antigen bindingmolecule to the activating T cell antigen, particularly CD3, withoutsimultaneous binding to HLA-A2/WT1 does not result in T cell activation.

In one embodiment, the bispecific antigen binding molecule is capable ofre-directing cytotoxic activity of a T cell to a target cell. In aparticular embodiment, said re-direction is independent of MHC-mediatedpeptide antigen presentation by the target cell and and/or specificityof the T cell. Particularly, a T cell according to any of theembodiments of the invention is a cytotoxic T cell. In some embodimentsthe T cell is a CD4⁺ or a CD8⁺ T cell, particularly a CD8⁺ T cell.

First Antigen Binding Moiety

The bispecific antigen binding molecule of the invention comprises atleast one antigen binding moiety, particularly a Fab molecule, thatbinds to HLA-A2/WT1 (first antigen). In certain embodiments, thebispecific antigen binding molecule comprises two antigen bindingmoieties, particularly Fab molecules, which bind to HLA-A2/WT1. In aparticular such embodiment, each of these antigen binding moieties bindsto the same antigenic determinant. In an even more particularembodiment, all of these antigen binding moieties are identical, i.e.they comprise the same amino acid sequences including the same aminoacid substitutions in the CH1 and CL domain as described herein (ifany). In one embodiment, the bispecific antigen binding moleculecomprises not more than two antigen binding moieties, particularly Fabmolecules, which bind to HLA-A2/WT1.

In particular embodiments, the antigen binding moiety(ies) which bind toHLA-A2/WT1 is/are a conventional Fab molecule. In such embodiments, theantigen binding moiety(ies) that binds to a second antigen is acrossover Fab molecule as described herein, i.e. a Fab molecule whereinthe variable domains VH and VL or the constant domains CH1 and CL of theFab heavy and light chains are exchanged/replaced by each other.

In alternative embodiments, the antigen binding moiety(ies) which bindto HLA-A2/WT1 is/are a crossover Fab molecule as described herein, i.e.a Fab molecule wherein the variable domains VH and VL or the constantdomains CH1 and CL of the Fab heavy and light chains areexchanged/replaced by each other. In such embodiments, the antigenbinding moiety(ies) that binds a second antigen is a conventional Fabmolecule.

The HLA-A2/WT1 binding moiety is able to direct the bispecific antigenbinding molecule to a target site, for example to a specific type oftumor cell that expresses HLA-A2/WT1.

The first antigen binding moiety of the bispecific antigen bindingmolecule may incorporate any of the features, singly or in combination,described herein in relation to the antibody that binds HLA-A2/WT1,unless scientifically clearly unreasonable or impossible.

Thus, in one aspect, the invention provides a bispecific antigen bindingmolecule, comprising (a) a first antigen binding moiety that binds to afirst antigen, wherein the first antigen is HLA-A2/WT1 and the firstantigen binding moiety comprises

(i) a heavy chain variable region (VH) comprising a heavy chaincomplementary determining region (HCDR) 1 of SEQ ID NO: 1, a HCDR 2 ofSEQ ID NO: 2, and a HCDR 3 of SEQ ID NO: 3, and a light chain variableregion (VL) comprising a light chain complementarity determining region(LCDR) 1 of SEQ ID NO: 4, a LCDR 2 of SEQ ID NO: 5 and a LCDR 3 of SEQID NO: 6;(ii) a VH comprising a HCDR 1 of SEQ ID NO: 9, a HCDR 2 of SEQ ID NO:10, and a HCDR 3 of SEQ ID NO: 11, and a VL comprising a LCDR 1 of SEQID NO: 12, a LCDR 2 of SEQ ID NO: 13 and a LCDR 3 of SEQ ID NO: 14;(iii) a VH comprising a HCDR 1 of SEQ ID NO: 17, a HCDR 2 of SEQ ID NO:18, and a HCDR 3 of SEQ ID NO: 19, and a VL comprising a LCDR 1 of SEQID NO: 20, a LCDR 2 of SEQ ID NO: 21 and a LCDR 3 of SEQ ID NO: 22;(iv) a VH comprising a HCDR 1 of SEQ ID NO: 25, a HCDR 2 of SEQ ID NO:26, and a HCDR 3 of SEQ ID NO: 27, and a VL comprising a LCDR 1 of SEQID NO: 28, a LCDR 2 of SEQ ID NO: 29 and a LCDR 3 of SEQ ID NO: 30;(v) a VH comprising a HCDR 1 of SEQ ID NO: 33, a HCDR 2 of SEQ ID NO:34, and a HCDR 3 of SEQ ID NO: 35, and a VL comprising a LCDR 1 of SEQID NO: 36, a LCDR 2 of SEQ ID NO: 37 and a LCDR 3 of SEQ ID NO: 38;(vi) a VH comprising a HCDR 1 of SEQ ID NO: 41, a HCDR 2 of SEQ ID NO:42, and a HCDR 3 of SEQ ID NO: 43, and a VL comprising a LCDR 1 of SEQID NO: 44, a LCDR 2 of SEQ ID NO: 45 and a LCDR 3 of SEQ ID NO: 46;(vii) a VH comprising a HCDR 1 of SEQ ID NO: 49, a HCDR 2 of SEQ ID NO:50, and a HCDR 3 of SEQ ID NO: 51, and a VL comprising a LCDR 1 of SEQID NO: 52, a LCDR 2 of SEQ ID NO: 53 and a LCDR 3 of SEQ ID NO: 54;(viii) a VH comprising a HCDR 1 of SEQ ID NO: 57, a HCDR 2 of SEQ ID NO:58, and a HCDR 3 of SEQ ID NO: 59, and a VL comprising a LCDR 1 of SEQID NO: 60, a LCDR 2 of SEQ ID NO: 61 and a LCDR 3 of SEQ ID NO: 62; or(ix) a VH comprising a HCDR 1 of SEQ ID NO: 65, a HCDR 2 of SEQ ID NO:66, and a HCDR 3 of SEQ ID NO: 67, and a VL comprising a LCDR 1 of SEQID NO: 68, a LCDR 2 of SEQ ID NO: 69 and a LCDR 3 of SEQ ID NO: 70, and(b) a second antigen binding moiety that binds to a second antigen.

In a particular embodiment, the first antigen binding moiety comprises aVH comprising a HCDR 1 of SEQ ID NO: 1, a HCDR 2 of SEQ ID NO: 2, and aHCDR 3 of SEQ ID NO: 3, and a VL comprising a LCDR 1 of SEQ ID NO: 4, aLCDR 2 of SEQ ID NO: 5 and a LCDR 3 of SEQ ID NO: 6.

In another embodiment, the first antigen binding moiety comprises a VHcomprising a HCDR 1 of SEQ ID NO: 9, a HCDR 2 of SEQ ID NO: 10, and aHCDR 3 of SEQ ID NO: 11, and a VL comprising a LCDR 1 of SEQ ID NO: 12,a LCDR 2 of SEQ ID NO: 13 and a LCDR 3 of SEQ ID NO: 14.

In a further embodiment, the first antigen binding moiety comprises a VHcomprising a HCDR 1 of SEQ ID NO: 17, a HCDR 2 of SEQ ID NO: 18, and aHCDR 3 of SEQ ID NO: 19, and a VL comprising a LCDR 1 of SEQ ID NO: 20,a LCDR 2 of SEQ ID NO: 21 and a LCDR 3 of SEQ ID NO: 22.

In still a further embodiment, the first antigen binding moietycomprises a VH comprising a HCDR 1 of SEQ ID NO: 25, a HCDR 2 of SEQ IDNO: 26, and a HCDR 3 of SEQ ID NO: 27, and a VL comprising a LCDR 1 ofSEQ ID NO: 28, a LCDR 2 of SEQ ID NO: 29 and a LCDR 3 of SEQ ID NO: 30.

In some embodiments, the first antigen binding moiety is (derived from)a human antibody. In one embodiment, the VH is a human VH and/or the VLis a human VL. In one embodiment, the first antigen binding moietycomprises CDRs as in any of the above embodiments, and further comprisesa human framework, e.g. a human immunoglobulin framework.

In one embodiment, the first antigen binding moiety comprises

(i) a VH comprising an amino acid sequence that is at least about 95%,96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQID NO: 7, and a VL comprising an amino acid sequence that is at leastabout 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acidsequence of SEQ ID NO: 8;(ii) a VH comprising an amino acid sequence that is at least about 95%,96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQID NO: 15, and a VL comprising an amino acid sequence that is at leastabout 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acidsequence of SEQ ID NO: 16;(iii) a VH comprising an amino acid sequence that is at least about 95%,96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQID NO: 23, and a VL comprising an amino acid sequence that is at leastabout 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acidsequence of SEQ ID NO: 24;(iv) a VH comprising an amino acid sequence that is at least about 95%,96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQID NO: 31, and a VL comprising an amino acid sequence that is at leastabout 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acidsequence of SEQ ID NO: 32;(v) a VH comprising an amino acid sequence that is at least about 95%,96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQID NO: 39, and a VL comprising an amino acid sequence that is at leastabout 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acidsequence of SEQ ID NO: 40;(vi) a VH comprising an amino acid sequence that is at least about 95%,96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQID NO: 47, and a VL comprising an amino acid sequence that is at leastabout 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acidsequence of SEQ ID NO: 48;(vii) a VH comprising an amino acid sequence that is at least about 95%,96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQID NO: 55, and a VL comprising an amino acid sequence that is at leastabout 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acidsequence of SEQ ID NO: 56;(viii) a VH comprising an amino acid sequence that is at least about95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence ofSEQ ID NO: 63, and a VL comprising an amino acid sequence that is atleast about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acidsequence of SEQ ID NO: 64; or(ix) a VH comprising an amino acid sequence that is at least about 95%,96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQID NO: 71, and a VL comprising an amino acid sequence that is at leastabout 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acidsequence of SEQ ID NO: 72.

In a particular embodiment, the first antigen binding moiety comprises aVH comprising an amino acid sequence that is at least about 95%, 96%,97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:7, and a VL comprising an amino acid sequence that is at least about95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence ofSEQ ID NO: 8.

In another embodiment, the first antigen binding moiety comprises a VHcomprising an amino acid sequence that is at least about 95%, 96%, 97%,98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 15,and a VL comprising an amino acid sequence that is at least about 95%,96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQID NO: 16.

In a further embodiment, the first antigen binding moiety comprises a VHcomprising an amino acid sequence that is at least about 95%, 96%, 97%,98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 23,and a VL comprising an amino acid sequence that is at least about 95%,96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQID NO: 24.

In still a further embodiment, the first antigen binding moietycomprises a VH comprising an amino acid sequence that is at least about95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence ofSEQ ID NO: 31, and a VL comprising an amino acid sequence that is atleast about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acidsequence of SEQ ID NO: 32. In one embodiment, the first antigen bindingmoiety comprises

(i) a VH sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO: 7, and a VL sequencethat is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to theamino acid sequence of SEQ ID NO: 8;(ii) a VH sequence that is at least about 95%, 96%, 97%, 98%, 99% or100% identical to the amino acid sequence of SEQ ID NO: 15, and a VLsequence that is at least about 95%, 96%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO: 16;(iii) a VH sequence that is at least about 95%, 96%, 97%, 98%, 99% or100% identical to the amino acid sequence of SEQ ID NO: 23, and a VLsequence that is at least about 95%, 96%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO: 24;(iv) a VH sequence that is at least about 95%, 96%, 97%, 98%, 99% or100% identical to the amino acid sequence of SEQ ID NO: 31, and a VLsequence that is at least about 95%, 96%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO: 32;(v) a VH sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO: 39, and a VL sequencethat is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to theamino acid sequence of SEQ ID NO: 40;(vi) a VH sequence that is at least about 95%, 96%, 97%, 98%, 99% or100% identical to the amino acid sequence of SEQ ID NO: 47, and a VLsequence that is at least about 95%, 96%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO: 48;(vii) a VH sequence that is at least about 95%, 96%, 97%, 98%, 99% or100% identical to the amino acid sequence of SEQ ID NO: 55, and a VLsequence that is at least about 95%, 96%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO: 56;(viii) a VH sequence that is at least about 95%, 96%, 97%, 98%, 99% or100% identical to the amino acid sequence of SEQ ID NO: 63, and a VLsequence that is at least about 95%, 96%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO: 64; or(ix) a VH sequence that is at least about 95%, 96%, 97%, 98%, 99% or100% identical to the amino acid sequence of SEQ ID NO: 71, and a VLsequence that is at least about 95%, 96%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO: 72.

In a particular embodiment, the first antigen binding moiety comprises aVH sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO: 7, and a VL sequencethat is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to theamino acid sequence of SEQ ID NO: 8.

In another embodiment, the first antigen binding moiety comprises a VHsequence that is at least about 95%, 96%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO: 15, and a VL sequencethat is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to theamino acid sequence of SEQ ID NO: 16.

In a further embodiment, the first antigen binding moiety comprises a VHsequence that is at least about 95%, 96%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO: 23, and a VL sequencethat is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to theamino acid sequence of SEQ ID NO: 24.

In still a further embodiment, the first antigen binding moietycomprises a VH sequence that is at least about 95%, 96%, 97%, 98%, 99%or 100% identical to the amino acid sequence of SEQ ID NO: 31, and a VLsequence that is at least about 95%, 96%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO: 32.

In one embodiment, the first antigen binding moiety comprises

(i) a VH comprising the amino acid sequence of SEQ ID NO: 7, and a VLcomprising the amino acid sequence of SEQ ID NO: 8;(ii) a VH comprising the amino acid sequence of SEQ ID NO: 15, and a VLcomprising the amino acid sequence of SEQ ID NO: 16;(iii) a VH comprising the amino acid sequence of SEQ ID NO: 23, and a VLcomprising the amino acid sequence of SEQ ID NO: 24;(iv) a VH comprising the amino acid sequence of SEQ ID NO: 31, and a VLcomprising the amino acid sequence of SEQ ID NO: 32;(v) a VH comprising the amino acid sequence of SEQ ID NO: 39, and a VLcomprising the amino acid sequence of SEQ ID NO: 40;(vi) a VH comprising the amino acid sequence of SEQ ID NO: 47, and a VLcomprising the amino acid sequence of SEQ ID NO: 48;(vii) a VH comprising the amino acid sequence of SEQ ID NO: 55, and a VLcomprising the amino acid sequence of SEQ ID NO: 56;(viii) a VH comprising the amino acid sequence of SEQ ID NO: 63, and aVL comprising the amino acid sequence of SEQ ID NO: 64; or(ix) a VH comprising the amino acid sequence of SEQ ID NO: 71, and a VLcomprising the amino acid sequence of SEQ ID NO: 72.

In one embodiment, the first antigen binding moiety comprises

(i) the VH sequence of SEQ ID NO: 7, and the VL sequence of SEQ ID NO:8;(ii) the VH sequence of SEQ ID NO: 15, and the VL sequence of SEQ ID NO:16;(iii) the VH sequence of SEQ ID NO: 23, and the VL sequence of SEQ IDNO: 24;(iv) the VH sequence of SEQ ID NO: 31, and the VL sequence of SEQ ID NO:32;(v) the VH sequence of SEQ ID NO: 39, and the VL sequence of SEQ ID NO:40;(vi) the VH sequence of SEQ ID NO: 47, and the VL sequence of SEQ ID NO:48;(vii) the VH sequence of SEQ ID NO: 55, and the VL sequence of SEQ IDNO: 56;(viii) the VH sequence of SEQ ID NO: 63, and the VL sequence of SEQ IDNO: 64; or(ix) the VH sequence of SEQ ID NO: 71, and the VL sequence of SEQ ID NO:72.

In a particular embodiment, the first antigen binding moiety comprises aVH comprising the amino acid sequence of SEQ ID NO: 7 and a VLcomprising the amino acid sequence of SEQ ID NO: 8.

In another embodiment, the first antigen binding moiety comprises a VHcomprising the amino acid sequence of SEQ ID NO: 15 and a VL comprisingthe amino acid sequence of SEQ ID NO: 16.

In a further embodiment, the first antigen binding moiety comprises a VHcomprising the amino acid sequence of SEQ ID NO: 23 and a VL comprisingthe amino acid sequence of SEQ ID NO: 24.

In still a further embodiment, the first antigen binding moietycomprises a VH comprising the amino acid sequence of SEQ ID NO: 31 and aVL comprising the amino acid sequence of SEQ ID NO: 32.

In a particular embodiment, the first antigen binding moiety comprisesthe VH sequence of SEQ ID NO: 7 and the VL sequence of SEQ ID NO: 8.

In another embodiment, the first antigen binding moiety comprises the VHsequence of SEQ ID NO: 15 and the VL sequence of SEQ ID NO: 16.

In a further embodiment, the first antigen binding moiety comprises theVH sequence of SEQ ID NO: 23 and the VL sequence of SEQ ID NO: 24.

In still a further embodiment, the first antigen binding moietycomprises the VH sequence of SEQ ID NO: 31 and the VL sequence of SEQ IDNO: 32.

In one embodiment, the first antigen binding moiety comprises a humanconstant region. In one embodiment, the first antigen binding moiety isa Fab molecule comprising a human constant region, particularly a humanCH1 and/or CL domain. Exemplary sequences of human constant domains aregiven in SEQ ID NOs 112 and 113 (human kappa and lambda CL domains,respectively) and SEQ ID NO: 114 (human IgG₁ heavy chain constantdomains CH1-CH2-CH3). In some embodiments, the first antigen bindingmoiety comprises a light chain constant region comprising an amino acidsequence that is at least about 95%, 96%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO: 112 or SEQ ID NO:113, particularly the amino acid sequence of SEQ ID NO: 112.Particularly, the light chain constant region may comprise amino acidmutations as described herein under “charge modifications” and/or maycomprise deletion or substitutions of one or more (particularly two)N-terminal amino acids if in a crossover Fab molecule. In someembodiments, the first antigen binding moiety comprises a heavy chainconstant region comprising an amino acid sequence that is at least about95%, 96%, 97%, 98%, 99% or 100% identical to the CH1 domain sequencecomprised in the amino acid sequence of SEQ ID NO: 114. Particularly,the heavy chain constant region (specifically CH1 domain) may compriseamino acid mutations as described herein under “charge modifications”.

Second Antigen Binding Moiety

The bispecific antigen binding molecule of the invention comprises atleast one antigen binding moiety, particularly a Fab molecule, thatbinds to a second antigen (different from HLA-A2/WT1). In particularembodiments, the antigen binding moiety that binds the second antigen isa crossover Fab molecule as described herein, i.e. a Fab moleculewherein the variable domains VH and VL or the constant domains CH1 andCL of the Fab heavy and light chains are exchanged/replaced by eachother. In such embodiments, the antigen binding moiety(ies) that bindsto the first antigen (i.e. HLA-A2/WT1) is preferably a conventional Fabmolecule. In embodiments where there is more than one antigen bindingmoiety, particularly Fab molecule, that binds to HLA-A2/WT1 comprised inthe bispecific antigen binding molecule, the antigen binding moiety thatbinds to the second antigen preferably is a crossover Fab molecule andthe antigen binding moieties that bind to HLA-A2/WT1 are conventionalFab molecules.

In alternative embodiments, the antigen binding moiety that binds to thesecond antigen is a conventional Fab molecule. In such embodiments, theantigen binding moiety(ies) that binds to the first antigen (i.e.HLA-A2/WT1) is a crossover Fab molecule as described herein, i.e. a Fabmolecule wherein the variable domains VH and VL or the constant domainsCH1 and CL of the Fab heavy and light chains are exchanged/replaced byeach other. In embodiments where there is more than one antigen bindingmoiety, particularly Fab molecule, that binds to a second antigencomprised in the bispecific antigen binding molecule, the antigenbinding moiety that binds to HLA-A2/WT1 preferably is a crossover Fabmolecule and the antigen binding moieties that bind to the secondantigen are conventional Fab molecules.

In some embodiments, the second antigen is an activating T cell antigen(also referred to herein as an “activating T cell antigen bindingmoiety, or activating T cell antigen binding Fab molecule”). In aparticular embodiment, the bispecific antigen binding molecule comprisesnot more than one antigen binding moiety capable of specific binding toan activating T cell antigen. In one embodiment the bispecific antigenbinding molecule provides monovalent binding to the activating T cellantigen.

In particular embodiments, the second antigen is CD3, particularly humanCD3 (SEQ ID NO: 107) or cynomolgus CD3 (SEQ ID NO: 108), mostparticularly human CD3. In one embodiment the second antigen bindingmoiety is cross-reactive for (i.e. specifically binds to) human andcynomolgus CD3. In some embodiments, the second antigen is the epsilonsubunit of CD3 (CD3 epsilon).

In one embodiment, the second antigen binding moiety comprises a HCDR 1of SEQ ID NO: 115, a HCDR 2 of SEQ ID NO: 116, a HCDR 3 of SEQ ID NO:117, a LCDR 1 of SEQ ID NO: 118, a LCDR 2 of SEQ ID NO: 119 and a LCDR 3of SEQ ID NO: 120.

In one embodiment, the second antigen binding moiety comprises a VHcomprising a HCDR 1 of SEQ ID NO: 115, a HCDR 2 of SEQ ID NO: 116, and aHCDR 3 of SEQ ID NO: 117, and a VL comprising a LCDR 1 of SEQ ID NO:118, a LCDR 2 of SEQ ID NO: 119 and a LCDR 3 of SEQ ID NO: 120.

In some embodiments, the second antigen binding moiety is (derived from)a humanized antibody.

In one embodiment, the VH is a humanized VH and/or the VL is a humanizedVL. In one embodiment, the second antigen binding moiety comprises CDRsas in any of the above embodiments, and further comprises an acceptorhuman framework, e.g. a human immunoglobulin framework or a humanconsensus framework.

In one embodiment, the second antigen binding moiety comprises a VHsequence that is at least about 95%, 96%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO: 121. In oneembodiment, the second antigen binding moiety comprises a VL sequencethat is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to theamino acid sequence of SEQ ID NO: 122.

In one embodiment, the second antigen binding moiety comprises a VHsequence that is at least about 95%, 96%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO: 121, and a VLsequence that is at least about 95%, 96%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO: 122.

In one embodiment, the VH of the second antigen binding moiety comprisesan amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or100% identical to the amino acid sequence of SEQ ID NO: 121, and the VLof the second antigen binding moiety comprises an amino acid sequencethat is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to theamino acid sequence of SEQ ID NO: 122.

In one embodiment, the second antigen binding moiety comprises a VHcomprising the amino acid sequence of SEQ ID NO: 121, and a VLcomprising the amino acid sequence of SEQ ID NO: 122. In one embodiment,the second antigen binding moiety comprises the VH sequence of SEQ IDNO: 121, and the VL sequence of SEQ ID NO: 122.

In a particular embodiment, the second antigen binding moiety comprisesa HCDR 1 of SEQ ID NO: 130, a HCDR 2 of SEQ ID NO: 131, a HCDR 3 of SEQID NO: 132, a LCDR 1 of SEQ ID NO: 133, a LCDR 2 of SEQ ID NO: 134 and aLCDR 3 of SEQ ID NO: 135.

In another particular embodiment, the second antigen binding moietycomprises a VH comprising a HCDR 1 of SEQ ID NO: 130, a HCDR 2 of SEQ IDNO: 131, and a HCDR 3 of SEQ ID NO: 132, and a VL comprising a LCDR 1 ofSEQ ID NO: 133, a LCDR 2 of SEQ ID NO: 134 and a LCDR 3 of SEQ ID NO:135.

In some embodiments, the second antigen binding moiety is (derived from)a humanized antibody.

In one embodiment, the VH is a humanized VH and/or the VL is a humanizedVL. In one embodiment, the second antigen binding moiety comprises CDRsas in any of the above embodiments, and further comprises an acceptorhuman framework, e.g. a human immunoglobulin framework or a humanconsensus framework.

In one embodiment, the second antigen binding moiety comprises a VHsequence that is at least about 95%, 96%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO: 136. In oneembodiment, the second antigen binding moiety comprises a VL sequencethat is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to theamino acid sequence of SEQ ID NO: 137.

In a particular embodiment, the second antigen binding moiety comprisesa VH sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO: 136, and a VLsequence that is at least about 95%, 96%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO: 137.

In another particular embodiment, the VH of the second antigen bindingmoiety comprises an amino acid sequence that is at least about 95%, 96%,97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:136, and the VL of the second antigen binding moiety comprises an aminoacid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO: 137.

In one embodiment, the second antigen binding moiety comprises a VHcomprising the amino acid sequence of SEQ ID NO: 136, and a VLcomprising the amino acid sequence of SEQ ID NO: 137.

In one embodiment, the second antigen binding moiety comprises the VHsequence of SEQ ID NO: 136, and the VL sequence of SEQ ID NO: 137.

In one embodiment, the second antigen binding moiety comprises a humanconstant region. In one embodiment, the second antigen binding moiety isa Fab molecule comprising a human constant region, particularly a humanCH1 and/or CL domain. Exemplary sequences of human constant domains aregiven in SEQ ID NOs 112 and 113 (human kappa and lambda CL domains,respectively) and SEQ ID NO: 114 (human IgG₁ heavy chain constantdomains CH1-CH2-CH3). In some embodiments, the second antigen bindingmoiety comprises a light chain constant region comprising an amino acidsequence that is at least about 95%, 96%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO: 112 or SEQ ID NO:113, particularly the amino acid sequence of SEQ ID NO: 112.Particularly, the light chain constant region may comprise amino acidmutations as described herein under “charge modifications” and/or maycomprise deletion or substitutions of one or more (particularly two)N-terminal amino acids if in a crossover Fab molecule. In someembodiments, the second antigen binding moiety comprises a heavy chainconstant region comprising an amino acid sequence that is at least about95%, 96%, 97%, 98%, 99% or 100% identical to the CH1 domain sequencecomprised in the amino acid sequence of SEQ ID NO: 114. Particularly,the heavy chain constant region (specifically CH1 domain) may compriseamino acid mutations as described herein under “charge modifications”.

In some embodiments, the second antigen binding moiety is a Fab moleculewherein the variable domains VL and VH or the constant domains CL andCH1, particularly the variable domains VL and VH, of the Fab light chainand the Fab heavy chain are replaced by each other (i.e. according tosuch embodiment, the second antigen binding moiety is a crossover Fabmolecule wherein the variable or constant domains of the Fab light chainand the Fab heavy chain are exchanged). In one such embodiment, thefirst (and the third, if any) antigen binding moiety is a conventionalFab molecule.

In one embodiment, not more than one antigen binding moiety that bindsto the second antigen (e.g. an activating T cell antigen such as CD3) ispresent in the bispecific antigen binding molecule (i.e. the bispecificantigen binding molecule provides monovalent binding to the secondantigen).

Charge Modifications

The bispecific antigen binding molecules of the invention may compriseamino acid substitutions in Fab molecules comprised therein which areparticularly efficient in reducing mispairing of light chains withnon-matching heavy chains (Bence-Jones-type side products), which canoccur in the production of Fab-based bi-/multispecific antigen bindingmolecules with a VH/VL exchange in one (or more, in case of moleculescomprising more than two antigen-binding Fab molecules) of their bindingarms (see also PCT publication no. WO 2015/150447, particularly theexamples therein, incorporated herein by reference in its entirety). Theratio of a desired bispecific antigen binding molecule compared toundesired side products, in particular Bence Jones-type side productsoccurring in bispecific antigen binding molecules with a VH/VL domainexchange in one of their binding arms, can be improved by theintroduction of charged amino acids with opposite charges at specificamino acid positions in the CH1 and CL domains (sometimes referred toherein as “charge modifications”).

Accordingly, in some embodiments wherein the first and the secondantigen binding moiety of the bispecific antigen binding molecule areboth Fab molecules, and in one of the antigen binding moieties(particularly the second antigen binding moiety) the variable domains VLand VH of the Fab light chain and the Fab heavy chain are replaced byeach other,

i) in the constant domain CL of the first antigen binding moiety theamino acid at position 124 is substituted by a positively charged aminoacid (numbering according to Kabat), and wherein in the constant domainCH1 of the first antigen binding moiety the amino acid at position 147or the amino acid at position 213 is substituted by a negatively chargedamino acid (numbering according to Kabat EU index); orii) in the constant domain CL of the second antigen binding moiety theamino acid at position 124 is substituted by a positively charged aminoacid (numbering according to Kabat), and wherein in the constant domainCH1 of the second antigen binding moiety the amino acid at position 147or the amino acid at position 213 is substituted by a negatively chargedamino acid (numbering according to Kabat EU index).

The bispecific antigen binding molecule does not comprise bothmodifications mentioned under i) and ii). The constant domains CL andCH1 of the antigen binding moiety having the VH/VL exchange are notreplaced by each other (i.e. remain unexchanged).

In a more specific embodiment,

i) in the constant domain CL of the first antigen binding moiety theamino acid at position 124 is substituted independently by lysine (K),arginine (R) or histidine (H) (numbering according to Kabat), and in theconstant domain CH1 of the first antigen binding moiety the amino acidat position 147 or the amino acid at position 213 is substitutedindependently by glutamic acid (E), or aspartic acid (D) (numberingaccording to Kabat EU index); orii) in the constant domain CL of the second antigen binding moiety theamino acid at position 124 is substituted independently by lysine (K),arginine (R) or histidine (H) (numbering according to Kabat), and in theconstant domain CH1 of the second antigen binding moiety the amino acidat position 147 or the amino acid at position 213 is substitutedindependently by glutamic acid (E), or aspartic acid (D) (numberingaccording to Kabat EU index).

In one such embodiment, in the constant domain CL of the first antigenbinding moiety the amino acid at position 124 is substitutedindependently by lysine (K), arginine (R) or histidine (H) (numberingaccording to Kabat), and in the constant domain CH1 of the first antigenbinding moiety the amino acid at position 147 or the amino acid atposition 213 is substituted independently by glutamic acid (E), oraspartic acid (D) (numbering according to Kabat EU index).

In a further embodiment, in the constant domain CL of the first antigenbinding moiety the amino acid at position 124 is substitutedindependently by lysine (K), arginine (R) or histidine (H) (numberingaccording to Kabat), and in the constant domain CH1 of the first antigenbinding moiety the amino acid at position 147 is substitutedindependently by glutamic acid (E), or aspartic acid (D) (numberingaccording to Kabat EU index).

In a particular embodiment, in the constant domain CL of the firstantigen binding moiety the amino acid at position 124 is substitutedindependently by lysine (K), arginine (R) or histidine (H) (numberingaccording to Kabat) and the amino acid at position 123 is substitutedindependently by lysine (K), arginine (R) or histidine (H) (numberingaccording to Kabat), and in the constant domain CH1 of the first antigenbinding moiety the amino acid at position 147 is substitutedindependently by glutamic acid (E), or aspartic acid (D) (numberingaccording to Kabat EU index) and the amino acid at position 213 issubstituted independently by glutamic acid (E), or aspartic acid (D)(numbering according to Kabat EU index).

In a more particular embodiment, in the constant domain CL of the firstantigen binding moiety the amino acid at position 124 is substituted bylysine (K) (numbering according to Kabat) and the amino acid at position123 is substituted by lysine (K) (numbering according to Kabat), and inthe constant domain CH1 of the first antigen binding moiety the aminoacid at position 147 is substituted by glutamic acid (E) (numberingaccording to Kabat EU index) and the amino acid at position 213 issubstituted by glutamic acid (E) (numbering according to Kabat EUindex).

In an even more particular embodiment, in the constant domain CL of thefirst antigen binding moiety the amino acid at position 124 issubstituted by lysine (K) (numbering according to Kabat) and the aminoacid at position 123 is substituted by arginine (R) (numbering accordingto Kabat), and in the constant domain CH1 of the first antigen bindingmoiety the amino acid at position 147 is substituted by glutamic acid(E) (numbering according to Kabat EU index) and the amino acid atposition 213 is substituted by glutamic acid (E) (numbering according toKabat EU index).

In particular embodiments, if amino acid substitutions according to theabove embodiments are made in the constant domain CL and the constantdomain CH1 of the first antigen binding moiety, the constant domain CLof the first antigen binding moiety is of kappa isotype.

Alternatively, the amino acid substitutions according to the aboveembodiments may be made in the constant domain CL and the constantdomain CH1 of the second antigen binding moiety instead of in theconstant domain CL and the constant domain CH1 of the first antigenbinding moiety. In particular such embodiments, the constant domain CLof the second antigen binding moiety is of kappa isotype.

Accordingly, in one embodiment, in the constant domain CL of the secondantigen binding moiety the amino acid at position 124 is substitutedindependently by lysine (K), arginine (R) or histidine (H) (numberingaccording to Kabat), and in the constant domain CH1 of the secondantigen binding moiety the amino acid at position 147 or the amino acidat position 213 is substituted independently by glutamic acid (E), oraspartic acid (D) (numbering according to Kabat EU index).

In a further embodiment, in the constant domain CL of the second antigenbinding moiety the amino acid at position 124 is substitutedindependently by lysine (K), arginine (R) or histidine (H) (numberingaccording to Kabat), and in the constant domain CH1 of the secondantigen binding moiety the amino acid at position 147 is substitutedindependently by glutamic acid (E), or aspartic acid (D) (numberingaccording to Kabat EU index).

In still another embodiment, in the constant domain CL of the secondantigen binding moiety the amino acid at position 124 is substitutedindependently by lysine (K), arginine (R) or histidine (H) (numberingaccording to Kabat) and the amino acid at position 123 is substitutedindependently by lysine (K), arginine (R) or histidine (H) (numberingaccording to Kabat), and in the constant domain CH1 of the secondantigen binding moiety the amino acid at position 147 is substitutedindependently by glutamic acid (E), or aspartic acid (D) (numberingaccording to Kabat EU index) and the amino acid at position 213 issubstituted independently by glutamic acid (E), or aspartic acid (D)(numbering according to Kabat EU index).

In one embodiment, in the constant domain CL of the second antigenbinding moiety the amino acid at position 124 is substituted by lysine(K) (numbering according to Kabat) and the amino acid at position 123 issubstituted by lysine (K) (numbering according to Kabat), and in theconstant domain CH1 of the second antigen binding moiety the amino acidat position 147 is substituted by glutamic acid (E) (numbering accordingto Kabat EU index) and the amino acid at position 213 is substituted byglutamic acid (E) (numbering according to Kabat EU index).

In another embodiment, in the constant domain CL of the second antigenbinding moiety the amino acid at position 124 is substituted by lysine(K) (numbering according to Kabat) and the amino acid at position 123 issubstituted by arginine (R) (numbering according to Kabat), and in theconstant domain CH1 of the second antigen binding moiety the amino acidat position 147 is substituted by glutamic acid (E) (numbering accordingto Kabat EU index) and the amino acid at position 213 is substituted byglutamic acid (E) (numbering according to Kabat EU index).

In a particular embodiment, the bispecific antigen binding molecule ofthe invention comprises

(a) a first antigen binding moiety that binds to a first antigen,wherein the first antigen is HLA-A2/WT1, specifically HLA-A2/WT1_(RMF),and the first antigen binding moiety is a Fab molecule comprising aheavy chain variable region (VH) comprising a heavy chain complementarydetermining region (HCDR) 1 of SEQ ID NO: 1, a HCDR 2 of SEQ ID NO: 2,and a HCDR 3 of SEQ ID NO: 3, and a light chain variable region (VL)comprising a light chain complementarity determining region (LCDR) 1 ofSEQ ID NO: 4, a LCDR 2 of SEQ ID NO: 5 and a LCDR 3 of SEQ ID NO: 6, and(b) a second antigen binding moiety that binds to a second antigen,wherein the second antigen binding moiety is a Fab molecule wherein thevariable domains VL and VH of the Fab light chain and the Fab heavychain are replaced by each other;wherein in the constant domain CL of the first antigen binding moietythe amino acid at position 124 is substituted independently by lysine(K), arginine (R) or histidine (H) (numbering according to Kabat) (in aparticular embodiment independently by lysine (K) or arginine (R)) andthe amino acid at position 123 is substituted independently by lysine(K), arginine (R) or histidine (H) (numbering according to Kabat) (in aparticular embodiment independently by lysine (K) or arginine (R)), andin the constant domain CH1 of the first antigen binding moiety the aminoacid at position 147 is substituted independently by glutamic acid (E),or aspartic acid (D) (numbering according to Kabat EU index) and theamino acid at position 213 is substituted independently by glutamic acid(E), or aspartic acid (D) (numbering according to Kabat EU index).

In another particular embodiment, the bispecific antigen bindingmolecule of the invention comprises

(a) a first antigen binding moiety that binds to a first antigen,wherein the first antigen is HLA-A2/WT1, specifically HLA-A2/WT1_(RMF),and the first antigen binding moiety is a Fab molecule comprising aheavy chain variable region (VH) comprising a heavy chain complementarydetermining region (HCDR) 1 of SEQ ID NO: 9, a HCDR 2 of SEQ ID NO: 10,and a HCDR 3 of SEQ ID NO: 11, and a light chain variable region (VL)comprising a light chain complementarity determining region (LCDR) 1 ofSEQ ID NO: 12, a LCDR 2 of SEQ ID NO: 13 and a LCDR 3 of SEQ ID NO: 14,and(b) a second antigen binding moiety that binds to a second antigen,wherein the second antigen binding moiety is a Fab molecule wherein thevariable domains VL and VH of the Fab light chain and the Fab heavychain are replaced by each other;wherein in the constant domain CL of the first antigen binding moietythe amino acid at position 124 is substituted independently by lysine(K), arginine (R) or histidine (H) (numbering according to Kabat) (in aparticular embodiment independently by lysine (K) or arginine (R)) andthe amino acid at position 123 is substituted independently by lysine(K), arginine (R) or histidine (H) (numbering according to Kabat) (in aparticular embodiment independently by lysine (K) or arginine (R)), andin the constant domain CH1 of the first antigen binding moiety the aminoacid at position 147 is substituted independently by glutamic acid (E),or aspartic acid (D) (numbering according to Kabat EU index) and theamino acid at position 213 is substituted independently by glutamic acid(E), or aspartic acid (D) (numbering according to Kabat EU index).

Bispecific Antigen Binding Molecule Formats

The components of the bispecific antigen binding molecule according tothe present invention can be fused to each other in a variety ofconfigurations. Exemplary configurations are depicted in FIG. 1.

In particular embodiments, the antigen binding moieties comprised in thebispecific antigen binding molecule are Fab molecules. In suchembodiments, the first, second, third etc. antigen binding moiety may bereferred to herein as first, second, third etc. Fab molecule,respectively.

In one embodiment, the first and the second antigen binding moiety ofthe bispecific antigen binding molecule are fused to each other,optionally via a peptide linker. In particular embodiments, the firstand the second antigen binding moiety are each a Fab molecule. In onesuch embodiment, the second antigen binding moiety is fused at theC-terminus of the Fab heavy chain to the N-terminus of the Fab heavychain of the first antigen binding moiety. In another such embodiment,the first antigen binding moiety is fused at the C-terminus of the Fabheavy chain to the N-terminus of the Fab heavy chain of the secondantigen binding moiety. In embodiments wherein either (i) the secondantigen binding moiety is fused at the C-terminus of the Fab heavy chainto the N-terminus of the Fab heavy chain of the first antigen bindingmoiety or (ii) the first antigen binding moiety is fused at theC-terminus of the Fab heavy chain to the N-terminus of the Fab heavychain of the second antigen binding moiety, additionally the Fab lightchain of the first antigen binding moiety and the Fab light chain of thesecond antigen binding moiety may be fused to each other, optionally viaa peptide linker.

A bispecific antigen binding molecule with a single antigen bindingmoiety (such as a Fab molecule) capable of specific binding to a targetcell antigen such as HLA-A2/WT1 (for example as shown in FIG. 1A, D, G,H, K, L) is useful, particularly in cases where internalization of thetarget cell antigen is to be expected following binding of a highaffinity antigen binding moiety. In such cases, the presence of morethan one antigen binding moiety specific for the target cell antigen mayenhance internalization of the target cell antigen, thereby reducing itsavailability.

In other cases, however, it will be advantageous to have a bispecificantigen binding molecule comprising two or more antigen binding moieties(such as Fab molecules) specific for a target cell antigen (see examplesshown in FIG. 1B, 1C, 1E, 1F, II, 1J, 1M or 1N), for example to optimizetargeting to the target site or to allow crosslinking of target cellantigens.

Accordingly, in particular embodiments, the bispecific antigen bindingmolecule according to the present invention comprises a third antigenbinding moiety.

In one embodiment, the third antigen binding moiety binds to the firstantigen, i.e. HLA-A2/WT1.

In one embodiment, the third antigen binding moiety is a Fab molecule.

In particular embodiments, the third antigen moiety is identical to thefirst antigen binding moiety.

The third antigen binding moiety of the bispecific antigen bindingmolecule may incorporate any of the features, singly or in combination,described herein in relation to the first antigen binding moiety and/orthe antibody that binds HLA-A2/WT1, unless scientifically clearlyunreasonable or impossible.

In one embodiment, the third antigen binding moiety comprises

(i) a heavy chain variable region (VH) comprising a heavy chaincomplementary determining region (HCDR) 1 of SEQ ID NO: 1, a HCDR 2 ofSEQ ID NO: 2, and a HCDR 3 of SEQ ID NO: 3, and a light chain variableregion (VL) comprising a light chain complementarity determining region(LCDR) 1 of SEQ ID NO: 4, a LCDR 2 of SEQ ID NO: 5 and a LCDR 3 of SEQID NO: 6;(ii) a VH comprising a HCDR 1 of SEQ ID NO: 9, a HCDR 2 of SEQ ID NO:10, and a HCDR 3 of SEQ ID NO: 11, and a VL comprising a LCDR 1 of SEQID NO: 12, a LCDR 2 of SEQ ID NO: 13 and a LCDR 3 of SEQ ID NO: 14;(iii) a VH comprising a HCDR 1 of SEQ ID NO: 17, a HCDR 2 of SEQ ID NO:18, and a HCDR 3 of SEQ ID NO: 19, and a VL comprising a LCDR 1 of SEQID NO: 20, a LCDR 2 of SEQ ID NO: 21 and a LCDR 3 of SEQ ID NO: 22;(iv) a VH comprising a HCDR 1 of SEQ ID NO: 25, a HCDR 2 of SEQ ID NO:26, and a HCDR 3 of SEQ ID NO: 27, and a VL comprising a LCDR 1 of SEQID NO: 28, a LCDR 2 of SEQ ID NO: 29 and a LCDR 3 of SEQ ID NO: 30;(v) a VH comprising a HCDR 1 of SEQ ID NO: 33, a HCDR 2 of SEQ ID NO:34, and a HCDR 3 of SEQ ID NO: 35, and a VL comprising a LCDR 1 of SEQID NO: 36, a LCDR 2 of SEQ ID NO: 37 and a LCDR 3 of SEQ ID NO: 38;(vi) a VH comprising a HCDR 1 of SEQ ID NO: 41, a HCDR 2 of SEQ ID NO:42, and a HCDR 3 of SEQ ID NO: 43, and a VL comprising a LCDR 1 of SEQID NO: 44, a LCDR 2 of SEQ ID NO: 45 and a LCDR 3 of SEQ ID NO: 46;(vii) a VH comprising a HCDR 1 of SEQ ID NO: 49, a HCDR 2 of SEQ ID NO:50, and a HCDR 3 of SEQ ID NO: 51, and a VL comprising a LCDR 1 of SEQID NO: 52, a LCDR 2 of SEQ ID NO: 53 and a LCDR 3 of SEQ ID NO: 54;(viii) a VH comprising a HCDR 1 of SEQ ID NO: 57, a HCDR 2 of SEQ ID NO:58, and a HCDR 3 of SEQ ID NO: 59, and a VL comprising a LCDR 1 of SEQID NO: 60, a LCDR 2 of SEQ ID NO: 61 and a LCDR 3 of SEQ ID NO: 62; or(ix) a VH comprising a HCDR 1 of SEQ ID NO: 65, a HCDR 2 of SEQ ID NO:66, and a HCDR 3 of SEQ ID NO: 67, and a VL comprising a LCDR 1 of SEQID NO: 68, a LCDR 2 of SEQ ID NO: 69 and a LCDR 3 of SEQ ID NO: 70.

In a particular embodiment, the third antigen binding moiety comprises aVH comprising a HCDR 1 of SEQ ID NO: 1, a HCDR 2 of SEQ ID NO: 2, and aHCDR 3 of SEQ ID NO: 3, and a VL comprising a LCDR 1 of SEQ ID NO: 4, aLCDR 2 of SEQ ID NO: 5 and a LCDR 3 of SEQ ID NO: 6.

In another embodiment, the third antigen binding moiety comprises a VHcomprising a HCDR 1 of SEQ ID NO: 9, a HCDR 2 of SEQ ID NO: 10, and aHCDR 3 of SEQ ID NO: 11, and a VL comprising a LCDR 1 of SEQ ID NO: 12,a LCDR 2 of SEQ ID NO: 13 and a LCDR 3 of SEQ ID NO: 14.

In a further embodiment, the third antigen binding moiety comprises a VHcomprising a HCDR 1 of SEQ ID NO: 17, a HCDR 2 of SEQ ID NO: 18, and aHCDR 3 of SEQ ID NO: 19, and a VL comprising a LCDR 1 of SEQ ID NO: 20,a LCDR 2 of SEQ ID NO: 21 and a LCDR 3 of SEQ ID NO: 22.

In still a further embodiment, the third antigen binding moietycomprises a VH comprising a HCDR 1 of SEQ ID NO: 25, a HCDR 2 of SEQ IDNO: 26, and a HCDR 3 of SEQ ID NO: 27, and a VL comprising a LCDR 1 ofSEQ ID NO: 28, a LCDR 2 of SEQ ID NO: 29 and a LCDR 3 of SEQ ID NO: 30.

In some embodiments, the third antigen binding moiety is (derived from)a human antibody. In one embodiment, the VH is a human VH and/or the VLis a human VL. In one embodiment, the third antigen binding moietycomprises CDRs as in any of the above embodiments, and further comprisesa human framework, e.g. a human immunoglobulin framework.

In one embodiment, the third antigen binding moiety comprises (i) a VHcomprising an amino acid sequence that is at least about 95%, 96%, 97%,98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 7,and a VL comprising an amino acid sequence that is at least about 95%,96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQID NO: 8;

(ii) a VH comprising an amino acid sequence that is at least about 95%,96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQID NO: 15, and a VL comprising an amino acid sequence that is at leastabout 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acidsequence of SEQ ID NO: 16;(iii) a VH comprising an amino acid sequence that is at least about 95%,96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQID NO: 23, and a VL comprising an amino acid sequence that is at leastabout 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acidsequence of SEQ ID NO: 24;(iv) a VH comprising an amino acid sequence that is at least about 95%,96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQID NO: 31, and a VL comprising an amino acid sequence that is at leastabout 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acidsequence of SEQ ID NO: 32;(v) a VH comprising an amino acid sequence that is at least about 95%,96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQID NO: 39, and a VL comprising an amino acid sequence that is at leastabout 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acidsequence of SEQ ID NO: 40;(vi) a VH comprising an amino acid sequence that is at least about 95%,96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQID NO: 47, and a VL comprising an amino acid sequence that is at leastabout 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acidsequence of SEQ ID NO: 48;(vii) a VH comprising an amino acid sequence that is at least about 95%,96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQID NO: 55, and a VL comprising an amino acid sequence that is at leastabout 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acidsequence of SEQ ID NO: 56;(viii) a VH comprising an amino acid sequence that is at least about95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence ofSEQ ID NO: 63, and a VL comprising an amino acid sequence that is atleast about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acidsequence of SEQ ID NO: 64; or(ix) a VH comprising an amino acid sequence that is at least about 95%,96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQID NO: 71, and a VL comprising an amino acid sequence that is at leastabout 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acidsequence of SEQ ID NO: 72.

In a particular embodiment, the third antigen binding moiety comprises aVH comprising an amino acid sequence that is at least about 95%, 96%,97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:7, and a VL comprising an amino acid sequence that is at least about95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence ofSEQ ID NO: 8.

In another embodiment, the third antigen binding moiety comprises a VHcomprising an amino acid sequence that is at least about 95%, 96%, 97%,98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 15,and a VL comprising an amino acid sequence that is at least about 95%,96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQID NO: 16.

In a further embodiment, the third antigen binding moiety comprises a VHcomprising an amino acid sequence that is at least about 95%, 96%, 97%,98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 23,and a VL comprising an amino acid sequence that is at least about 95%,96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQID NO: 24.

In still a further embodiment, the third antigen binding moietycomprises a VH comprising an amino acid sequence that is at least about95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence ofSEQ ID NO: 31, and a VL comprising an amino acid sequence that is atleast about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acidsequence of SEQ ID NO: 32.

In one embodiment, the third antigen binding moiety comprises

(i) a VH sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO: 7, and a VL sequencethat is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to theamino acid sequence of SEQ ID NO: 8;(ii) a VH sequence that is at least about 95%, 96%, 97%, 98%, 99% or100% identical to the amino acid sequence of SEQ ID NO: 15, and a VLsequence that is at least about 95%, 96%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO: 16;(iii) a VH sequence that is at least about 95%, 96%, 97%, 98%, 99% or100% identical to the amino acid sequence of SEQ ID NO: 23, and a VLsequence that is at least about 95%, 96%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO: 24;(iv) a VH sequence that is at least about 95%, 96%, 97%, 98%, 99% or100% identical to the amino acid sequence of SEQ ID NO: 31, and a VLsequence that is at least about 95%, 96%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO: 32;(v) a VH sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO: 39, and a VL sequencethat is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to theamino acid sequence of SEQ ID NO: 40;(vi) a VH sequence that is at least about 95%, 96%, 97%, 98%, 99% or100% identical to the amino acid sequence of SEQ ID NO: 47, and a VLsequence that is at least about 95%, 96%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO: 48;(vii) a VH sequence that is at least about 95%, 96%, 97%, 98%, 99% or100% identical to the amino acid sequence of SEQ ID NO: 55, and a VLsequence that is at least about 95%, 96%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO: 56;(viii) a VH sequence that is at least about 95%, 96%, 97%, 98%, 99% or100% identical to the amino acid sequence of SEQ ID NO: 63, and a VLsequence that is at least about 95%, 96%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO: 64; or(ix) a VH sequence that is at least about 95%, 96%, 97%, 98%, 99% or100% identical to the amino acid sequence of SEQ ID NO: 71, and a VLsequence that is at least about 95%, 96%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO: 72.

In a particular embodiment, the third antigen binding moiety comprises aVH sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO: 7, and a VL sequencethat is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to theamino acid sequence of SEQ ID NO: 8.

In another embodiment, the third antigen binding moiety comprises a VHsequence that is at least about 95%, 96%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO: 15, and a VL sequencethat is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to theamino acid sequence of SEQ ID NO: 16.

In a further embodiment, the third antigen binding moiety comprises a VHsequence that is at least about 95%, 96%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO: 23, and a VL sequencethat is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to theamino acid sequence of SEQ ID NO: 24.

In still a further embodiment, the third antigen binding moietycomprises a VH sequence that is at least about 95%, 96%, 97%, 98%, 99%or 100% identical to the amino acid sequence of SEQ ID NO: 31, and a VLsequence that is at least about 95%, 96%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO: 32.

In one embodiment, the third antigen binding moiety comprises

(i) a VH comprising the amino acid sequence of SEQ ID NO: 7, and a VLcomprising the amino acid sequence of SEQ ID NO: 8;(ii) a VH comprising the amino acid sequence of SEQ ID NO: 15, and a VLcomprising the amino acid sequence of SEQ ID NO: 16;(iii) a VH comprising the amino acid sequence of SEQ ID NO: 23, and a VLcomprising the amino acid sequence of SEQ ID NO: 24;(iv) a VH comprising the amino acid sequence of SEQ ID NO: 31, and a VLcomprising the amino acid sequence of SEQ ID NO: 32;(v) a VH comprising the amino acid sequence of SEQ ID NO: 39, and a VLcomprising the amino acid sequence of SEQ ID NO: 40;(vi) a VH comprising the amino acid sequence of SEQ ID NO: 47, and a VLcomprising the amino acid sequence of SEQ ID NO: 48;(vii) a VH comprising the amino acid sequence of SEQ ID NO: 55, and a VLcomprising the amino acid sequence of SEQ ID NO: 56;(viii) a VH comprising the amino acid sequence of SEQ ID NO: 63, and aVL comprising the amino acid sequence of SEQ ID NO: 64; or(ix) a VH comprising the amino acid sequence of SEQ ID NO: 71, and a VLcomprising the amino acid sequence of SEQ ID NO: 72.

In one embodiment, the third antigen binding moiety comprises

(i) the VH sequence of SEQ ID NO: 7, and the VL sequence of SEQ ID NO:8;(ii) the VH sequence of SEQ ID NO: 15, and the VL sequence of SEQ ID NO:16;(iii) the VH sequence of SEQ ID NO: 23, and the VL sequence of SEQ IDNO: 24;(iv) the VH sequence of SEQ ID NO: 31, and the VL sequence of SEQ ID NO:32;(v) the VH sequence of SEQ ID NO: 39, and the VL sequence of SEQ ID NO:40;(vi) the VH sequence of SEQ ID NO: 47, and the VL sequence of SEQ ID NO:48;(vii) the VH sequence of SEQ ID NO: 55, and the VL sequence of SEQ IDNO: 56;(viii) the VH sequence of SEQ ID NO: 63, and the VL sequence of SEQ IDNO: 64; or(ix) the VH sequence of SEQ ID NO: 71, and the VL sequence of SEQ ID NO:72.

In a particular embodiment, the third antigen binding moiety comprises aVH comprising the amino acid sequence of SEQ ID NO: 7 and a VLcomprising the amino acid sequence of SEQ ID NO: 8.

In another embodiment, the third antigen binding moiety comprises a VHcomprising the amino acid sequence of SEQ ID NO: 15 and a VL comprisingthe amino acid sequence of SEQ ID NO: 16.

In a further embodiment, the third antigen binding moiety comprises a VHcomprising the amino acid sequence of SEQ ID NO: 23 and a VL comprisingthe amino acid sequence of SEQ ID NO: 24.

In still a further embodiment, the third antigen binding moietycomprises a VH comprising the amino acid sequence of SEQ ID NO: 31 and aVL comprising the amino acid sequence of SEQ ID NO: 32.

In a particular embodiment, the third antigen binding moiety comprisesthe VH sequence of SEQ ID NO: 7 and the VL sequence of SEQ ID NO: 8.

In another embodiment, the third antigen binding moiety comprises the VHsequence of SEQ ID NO: 15 and the VL sequence of SEQ ID NO: 16.

In a further embodiment, the third antigen binding moiety comprises theVH sequence of SEQ ID NO: 23 and the VL sequence of SEQ ID NO: 24.

In still a further embodiment, the third antigen binding moietycomprises the VH sequence of SEQ ID NO: 31 and the VL sequence of SEQ IDNO: 32.

In one embodiment, the third antigen binding moiety comprises a humanconstant region. In one embodiment, the third antigen binding moiety isa Fab molecule comprising a human constant region, particularly a humanCH1 and/or CL domain. Exemplary sequences of human constant domains aregiven in SEQ ID NOs 112 and 113 (human kappa and lambda CL domains,respectively) and SEQ ID NO: 114 (human IgG₁ heavy chain constantdomains CH1-CH2-CH3).

In some embodiments, the third antigen binding moiety comprises a lightchain constant region comprising an amino acid sequence that is at leastabout 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acidsequence of SEQ ID NO: 112 or SEQ ID NO: 113, particularly the aminoacid sequence of SEQ ID NO: 112. Particularly, the light chain constantregion may comprise amino acid mutations as described herein under“charge modifications” and/or may comprise deletion or substitutions ofone or more (particularly two) N-terminal amino acids if in a crossoverFab molecule. In some embodiments, the third antigen binding moietycomprises a heavy chain constant region comprising an amino acidsequence that is at least about 95%, 96%, 97%, 98%, 99% or 100%identical to the CH1 domain sequence comprised in the amino acidsequence of SEQ ID NO: 114. Particularly, the heavy chain constantregion (specifically CH1 domain) may comprise amino acid mutations asdescribed herein under “charge modifications”.

In particular embodiments, the third and the first antigen bindingmoiety are each a Fab molecule and the third antigen binding moiety isidentical to the first antigen binding moiety. Thus, in theseembodiments the first and the third antigen binding moiety comprise thesame heavy and light chain amino acid sequences and have the samearrangement of domains (i.e. conventional or crossover)). Furthermore,in these embodiments, the third antigen binding moiety comprises thesame amino acid substitutions, if any, as the first antigen bindingmoiety. For example, the amino acid substitutions described herein as“charge modifications” will be made in the constant domain CL and theconstant domain CH1 of each of the first antigen binding moiety and thethird antigen binding moiety. Alternatively, said amino acidsubstitutions may be made in the constant domain CL and the constantdomain CH1 of the second antigen binding moiety (which in particularembodiments is also a Fab molecule), but not in the constant domain CLand the constant domain CH1 of the first antigen binding moiety and thethird antigen binding moiety.

Like the first antigen binding moiety, the third antigen binding moietyparticularly is a conventional Fab molecule. Embodiments wherein thefirst and the third antigen binding moieties are crossover Fab molecules(and the second antigen binding moiety is a conventional Fab molecule)are, however, also contemplated. Thus, in particular embodiments, thefirst and the third antigen binding moieties are each a conventional Fabmolecule, and the second antigen binding moiety is a crossover Fabmolecule as described herein, i.e. a Fab molecule wherein the variabledomains VH and VL or the constant domains CL and CH1 of the Fab heavyand light chains are exchanged/replaced by each other. In otherembodiments, the first and the third antigen binding moieties are each acrossover Fab molecule and the second antigen binding moiety is aconventional Fab molecule.

If a third antigen binding moiety is present, in a particular embodimentthe first and the third antigen moiety bind to HLA-A2/WT1, and thesecond antigen binding moiety binds to a second antigen, particularly anactivating T cell antigen, more particularly CD3, most particularly CD3epsilon.

In particular embodiments, the bispecific antigen binding moleculecomprises an Fc domain composed of a first and a second subunit. Thefirst and the second subunit of the Fc domain are capable of stableassociation.

The bispecific antigen binding molecule according to the invention canhave different configurations, i.e. the first, second (and optionallythird) antigen binding moiety may be fused to each other and to the Fcdomain in different ways. The components may be fused to each otherdirectly or, preferably, via one or more suitable peptide linkers. Wherefusion of a Fab molecule is to the N-terminus of a subunit of the Fcdomain, it is typically via an immunoglobulin hinge region.

In some embodiments, the first and the second antigen binding moiety areeach a Fab molecule and the second antigen binding moiety is fused atthe C-terminus of the Fab heavy chain to the N-terminus of the first orthe second subunit of the Fc domain. In such embodiments, the firstantigen binding moiety may be fused at the C-terminus of the Fab heavychain to the N-terminus of the Fab heavy chain of the second antigenbinding moiety or to the N-terminus of the other one of the subunits ofthe Fc domain. In particular such embodiments, said first antigenbinding moiety is a conventional Fab molecule, and the second antigenbinding moiety is a crossover Fab molecule as described herein, i.e. aFab molecule wherein the variable domains VH and VL or the constantdomains CL and CH1 of the Fab heavy and light chains areexchanged/replaced by each other. In other such embodiments, said firstFab molecule is a crossover Fab molecule and the second Fab molecule isa conventional Fab molecule.

In one embodiment, the first and the second antigen binding moiety areeach a Fab molecule, the second antigen binding moiety is fused at theC-terminus of the Fab heavy chain to the N-terminus of the first or thesecond subunit of the Fc domain, and the first antigen binding moiety isfused at the C-terminus of the Fab heavy chain to the N-terminus of theFab heavy chain of the second antigen binding moiety. In a specificembodiment, the bispecific antigen binding molecule essentially consistsof the first and the second Fab molecule, the Fc domain composed of afirst and a second subunit, and optionally one or more peptide linkers,wherein the first Fab molecule is fused at the C-terminus of the Fabheavy chain to the N-terminus of the Fab heavy chain of the second Fabmolecule, and the second Fab molecule is fused at the C-terminus of theFab heavy chain to the N-terminus of the first or the second subunit ofthe Fc domain. Such a configuration is schematically depicted in FIGS.1G and 1K (with the second antigen binding domain in these examplesbeing a VH/VL crossover Fab molecule). Optionally, the Fab light chainof the first Fab molecule and the Fab light chain of the second Fabmolecule may additionally be fused to each other.

In another embodiment, the first and the second antigen binding moietyare each a Fab molecule and the first and the second antigen bindingmoiety are each fused at the C-terminus of the Fab heavy chain to theN-terminus of one of the subunits of the Fc domain. In a specificembodiment, the bispecific antigen binding molecule essentially consistsof the first and the second Fab molecule, the Fc domain composed of afirst and a second subunit, and optionally one or more peptide linkers,wherein the first and the second Fab molecule are each fused at theC-terminus of the Fab heavy chain to the N-terminus of one of thesubunits of the Fc domain. Such a configuration is schematicallydepicted in FIGS. 1A and 1D (in these examples with the second antigenbinding domain being a VH/VL crossover Fab molecule and the firstantigen binding moiety being a conventional Fab molecule). The first andthe second Fab molecule may be fused to the Fc domain directly orthrough a peptide linker. In a particular embodiment the first and thesecond Fab molecule are each fused to the Fc domain through animmunoglobulin hinge region.

In a specific embodiment, the immunoglobulin hinge region is a humanIgG₁ hinge region, particularly where the Fc domain is an IgG₁ Fcdomain.

In some embodiments, the first and the second antigen binding moiety areeach a Fab molecule and the first antigen binding moiety is fused at theC-terminus of the Fab heavy chain to the N-terminus of the first or thesecond subunit of the Fc domain. In such embodiments, the second antigenbinding moiety may be fused at the C-terminus of the Fab heavy chain tothe N-terminus of the Fab heavy chain of the second antigen bindingmoiety or (as described above) to the N-terminus of the other one of thesubunits of the Fc domain. In particular such embodiments, said firstantigen binding moiety is a conventional Fab molecule, and the secondantigen binding moiety is a crossover Fab molecule as described herein,i.e. a Fab molecule wherein the variable domains VH and VL or theconstant domains CL and CH1 of the Fab heavy and light chains areexchanged/replaced by each other. In other such embodiments, said firstFab molecule is a crossover Fab molecule and the second Fab molecule isa conventional Fab molecule.

In one embodiment, the first and the second antigen binding moiety areeach a Fab molecule, the first antigen binding moiety is fused at theC-terminus of the Fab heavy chain to the N-terminus of the first or thesecond subunit of the Fc domain, and the second antigen binding moietyis fused at the C-terminus of the Fab heavy chain to the N-terminus ofthe Fab heavy chain of the first antigen binding moiety. In a specificembodiment, the bispecific antigen binding molecule essentially consistsof the first and the second Fab molecule, the Fc domain composed of afirst and a second subunit, and optionally one or more peptide linkers,wherein the second Fab molecule is fused at the C-terminus of the Fabheavy chain to the N-terminus of the Fab heavy chain of the first Fabmolecule, and the first Fab molecule is fused at the C-terminus of theFab heavy chain to the N-terminus of the first or the second subunit ofthe Fc domain. Such a configuration is schematically depicted in FIGS.1H and 1L (in these examples with the second antigen binding domainbeing a VH/VL crossover Fab molecule and the first antigen bindingmoiety being a conventional Fab molecule). Optionally, the Fab lightchain of the first Fab molecule and the Fab light chain of the secondFab molecule may additionally be fused to each other.

In some embodiments, a third antigen binding moiety, particularly athird Fab molecule, is fused at the C-terminus of the Fab heavy chain tothe N-terminus of the first or second subunit of the Fc domain. Inparticular such embodiments, said first and third Fab molecules are eacha conventional Fab molecule, and the second Fab molecule is a crossoverFab molecule as described herein, i.e. a Fab molecule wherein thevariable domains VH and VL or the constant domains CL and CH1 of the Fabheavy and light chains are exchanged/replaced by each other. In othersuch embodiments, said first and third Fab molecules are each acrossover Fab molecule and the second Fab molecule is a conventional Fabmolecule.

In a particular such embodiment, the second and the third antigenbinding moiety are each fused at the C-terminus of the Fab heavy chainto the N-terminus of one of the subunits of the Fc domain, and the firstantigen binding moiety is fused at the C-terminus of the Fab heavy chainto the N-terminus of the Fab heavy chain of the second Fab molecule. Ina specific embodiment, the bispecific antigen binding moleculeessentially consists of the first, the second and the third Fabmolecule, the Fc domain composed of a first and a second subunit, andoptionally one or more peptide linkers, wherein the first Fab moleculeis fused at the C-terminus of the Fab heavy chain to the N-terminus ofthe Fab heavy chain of the second Fab molecule, and the second Fabmolecule is fused at the C-terminus of the Fab heavy chain to theN-terminus of the first subunit of the Fc domain, and wherein the thirdFab molecule is fused at the C-terminus of the Fab heavy chain to theN-terminus of the second subunit of the Fc domain. Such a configurationis schematically depicted in FIGS. 1B and 1E (in these examples with thesecond antigen binding moiety being a VH/VL crossover Fab molecule, andthe first and the third antigen binding moiety being a conventional Fabmolecule), and FIGS. 1J and 1N (in these examples with the secondantigen binding moiety being a conventional Fab molecule, and the firstand the third antigen binding moiety being a VH/VL crossover Fabmolecule). The second and the third Fab molecule may be fused to the Fcdomain directly or through a peptide linker. In a particular embodimentthe second and the third Fab molecule are each fused to the Fc domainthrough an immunoglobulin hinge region. In a specific embodiment, theimmunoglobulin hinge region is a human IgG₁ hinge region, particularlywhere the Fc domain is an IgG₁ Fc domain. Optionally, the Fab lightchain of the first Fab molecule and the Fab light chain of the secondFab molecule may additionally be fused to each other.

In another such embodiment, the first and the third antigen bindingmoiety are each fused at the C-terminus of the Fab heavy chain to theN-terminus of one of the subunits of the Fc domain, and the secondantigen binding moiety is fused at the C-terminus of the Fab heavy chainto the N-terminus of the Fab heavy chain of the first antigen bindingmoiety. In a specific embodiment, the bispecific antigen bindingmolecule essentially consists of the first, the second and the third Fabmolecule, the Fc domain composed of a first and a second subunit, andoptionally one or more peptide linkers, wherein the second Fab moleculeis fused at the C-terminus of the Fab heavy chain to the N-terminus ofthe Fab heavy chain of the first Fab molecule, and the first Fabmolecule is fused at the C-terminus of the Fab heavy chain to theN-terminus of the first subunit of the Fc domain, and wherein the thirdFab molecule is fused at the C-terminus of the Fab heavy chain to theN-terminus of the second subunit of the Fc domain. Such a configurationis schematically depicted in FIGS. 1C and 1F (in these examples with thesecond antigen binding moiety being a VH/VL crossover Fab molecule, andthe first and the third antigen binding moiety being a conventional Fabmolecule) and in FIGS. 1I and 1M (in these examples with the secondantigen binding moiety being a conventional Fab molecule, and the firstand the third antigen binding moiety being a VH/VL crossover Fabmolecule). The first and the third Fab molecule may be fused to the Fcdomain directly or through a peptide linker. In a particular embodimentthe first and the third Fab molecule are each fused to the Fc domainthrough an immunoglobulin hinge region. In a specific embodiment, theimmunoglobulin hinge region is a human IgG₁ hinge region, particularlywhere the Fc domain is an IgG₁ Fc domain. Optionally, the Fab lightchain of the first Fab molecule and the Fab light chain of the secondFab molecule may additionally be fused to each other.

In configurations of the bispecific antigen binding molecule wherein aFab molecule is fused at the C-terminus of the Fab heavy chain to theN-terminus of each of the subunits of the Fc domain through animmunoglobulin hinge regions, the two Fab molecules, the hinge regionsand the Fc domain essentially form an immunoglobulin molecule. In aparticular embodiment the immunoglobulin molecule is an IgG classimmunoglobulin. In an even more particular embodiment the immunoglobulinis an IgG₁ subclass immunoglobulin. In another embodiment theimmunoglobulin is an IgG₄ subclass immunoglobulin. In a furtherparticular embodiment the immunoglobulin is a human immunoglobulin. Inother embodiments the immunoglobulin is a chimeric immunoglobulin or ahumanized immunoglobulin. In one embodiment, the immunoglobulincomprises a human constant region, particularly a human Fc region.

In some of the bispecific antigen binding molecule of the invention, theFab light chain of the first Fab molecule and the Fab light chain of thesecond Fab molecule are fused to each other, optionally via a peptidelinker. Depending on the configuration of the first and the second Fabmolecule, the Fab light chain of the first Fab molecule may be fused atits C-terminus to the N-terminus of the Fab light chain of the secondFab molecule, or the Fab light chain of the second Fab molecule may befused at its C-terminus to the N-terminus of the Fab light chain of thefirst Fab molecule. Fusion of the Fab light chains of the first and thesecond Fab molecule further reduces mispairing of unmatched Fab heavyand light chains, and also reduces the number of plasmids needed forexpression of some of the bispecific antigen binding molecules of theinvention.

The antigen binding moieties may be fused to the Fc domain or to eachother directly or through a peptide linker, comprising one or more aminoacids, typically about 2-20 amino acids. Peptide linkers are known inthe art and are described herein. Suitable, non-immunogenic peptidelinkers include, for example, (G₄S)_(n), (SG₄)_(n), (G₄S)_(n) orG₄(SG₄)_(n) peptide linkers. “n” is generally an integer from 1 to 10,typically from 2 to 4. In one embodiment said peptide linker has alength of at least 5 amino acids, in one embodiment a length of 5 to100, in a further embodiment of 10 to 50 amino acids. In one embodimentsaid peptide linker is (GxS)_(n) or (GxS)_(n)G_(m) with G=glycine,S=serine, and (x=3, n=3, 4, 5 or 6, and m=0, 1, 2 or 3) or (x=4, n=2, 3,4 or 5 and m=0, 1, 2 or 3), in one embodiment x=4 and n=2 or 3, in afurther embodiment x=4 and n=2. In one embodiment said peptide linker is(G45)₂. A particularly suitable peptide linker for fusing the Fab lightchains of the first and the second Fab molecule to each other is (G₄S)₂.An exemplary peptide linker suitable for connecting the Fab heavy chainsof the first and the second Fab fragments comprises the sequence(D)-(G₄S)₂ (SEQ ID NOs 110 and 111). Another suitable such linkercomprises the sequence (G₄S)₄. Additionally, linkers may comprise (aportion of) an immunoglobulin hinge region. Particularly where a Fabmolecule is fused to the N-terminus of an Fc domain subunit, it may befused via an immunoglobulin hinge region or a portion thereof, with orwithout an additional peptide linker.

In certain embodiments the bispecific antigen binding molecule accordingto the invention comprises a polypeptide wherein the Fab light chainvariable region of the second Fab molecule shares a carboxy-terminalpeptide bond with the Fab heavy chain constant region of the second Fabmolecule (i.e. the second Fab molecule comprises a crossover Fab heavychain, wherein the heavy chain variable region is replaced by a lightchain variable region), which in turn shares a carboxy-terminal peptidebond with an Fc domain subunit (VL₍₂₎-CH1₍₂₎-CH2-CH3(-CH4)), and apolypeptide wherein the Fab heavy chain of the first Fab molecule sharesa carboxy-terminal peptide bond with an Fc domain subunit(VH₍₁₎-CH1₍₁₎-CH2-CH3(-CH4)). In some embodiments the bispecific antigenbinding molecule further comprises a polypeptide wherein the Fab heavychain variable region of the second Fab molecule shares acarboxy-terminal peptide bond with the Fab light chain constant regionof the second Fab molecule (VH₍₂₎-CL₍₂₎) and the Fab light chainpolypeptide of the first Fab molecule (VL₍₁₎-CL₍₁₎). In certainembodiments the polypeptides are covalently linked, e.g., by a disulfidebond.

In certain embodiments the bispecific antigen binding molecule accordingto the invention comprises a polypeptide wherein the Fab heavy chainvariable region of the second Fab molecule shares a carboxy-terminalpeptide bond with the Fab light chain constant region of the second Fabmolecule (i.e. the second Fab molecule comprises a crossover Fab heavychain, wherein the heavy chain constant region is replaced by a lightchain constant region), which in turn shares a carboxy-terminal peptidebond with an Fc domain subunit (VH₍₂₎-CL₍₂₎-CH2-CH3(-CH4)), and apolypeptide wherein the Fab heavy chain of the first Fab molecule sharesa carboxy-terminal peptide bond with an Fc domain subunit(VH₍₁₎-CH1₍₁₎-CH2-CH3(-CH4)). In some embodiments the bispecific antigenbinding molecule further comprises a polypeptide wherein the Fab lightchain variable region of the second Fab molecule shares acarboxy-terminal peptide bond with the Fab heavy chain constant regionof the second Fab molecule (VL₍₂₎-CH1₍₂₎) and the Fab light chainpolypeptide of the first Fab molecule (VL₍₁₎-CL₍₁₎). In certainembodiments the polypeptides are covalently linked, e.g., by a disulfidebond.

In some embodiments, the bispecific antigen binding molecule comprises apolypeptide wherein the Fab light chain variable region of the secondFab molecule shares a carboxy-terminal peptide bond with the Fab heavychain constant region of the second Fab molecule (i.e. the second Fabmolecule comprises a crossover Fab heavy chain, wherein the heavy chainvariable region is replaced by a light chain variable region), which inturn shares a carboxy-terminal peptide bond with the Fab heavy chain ofthe first Fab molecule, which in turn shares a carboxy-terminal peptidebond with an Fc domain subunit(VL₍₂₎-CH1₍₂₎-VH₍₁₎-CH1₍₁₎-CH2-CH3(-CH4)). In other embodiments, thebispecific antigen binding molecule comprises a polypeptide wherein theFab heavy chain of the first Fab molecule shares a carboxy-terminalpeptide bond with the Fab light chain variable region of the second Fabmolecule which in turn shares a carboxy-terminal peptide bond with theFab heavy chain constant region of the second Fab molecule (i.e. thesecond Fab molecule comprises a crossover Fab heavy chain, wherein theheavy chain variable region is replaced by a light chain variableregion), which in turn shares a carboxy-terminal peptide bond with an Fcdomain subunit (VH₍₁₎-CH1₍₁₎-VL₍₂₎-CH1₍₂₎-CH2-CH3(-CH4)).

In some of these embodiments the bispecific antigen binding moleculefurther comprises a crossover Fab light chain polypeptide of the secondFab molecule, wherein the Fab heavy chain variable region of the secondFab molecule shares a carboxy-terminal peptide bond with the Fab lightchain constant region of the second Fab molecule (VH₍₂₎-CL₍₂₎), and theFab light chain polypeptide of the first Fab molecule (VL₍₁₎-CL₍₁₎). Inothers of these embodiments the bispecific antigen binding moleculefurther comprises a polypeptide wherein the Fab heavy chain variableregion of the second Fab molecule shares a carboxy-terminal peptide bondwith the Fab light chain constant region of the second Fab moleculewhich in turn shares a carboxy-terminal peptide bond with the Fab lightchain polypeptide of the first Fab molecule (VH₍₂₎-CL₍₂₎-VL₍₁₎-CL₍₁₎),or a polypeptide wherein the Fab light chain polypeptide of the firstFab molecule shares a carboxy-terminal peptide bond with the Fab heavychain variable region of the second Fab molecule which in turn shares acarboxy-terminal peptide bond with the Fab light chain constant regionof the second Fab molecule (VL₍₁₎-CL₍₁₎-VH₍₂₎-CL₍₂₎), as appropriate.

The bispecific antigen binding molecule according to these embodimentsmay further comprise (i) an Fc domain subunit polypeptide(CH2-CH3(-CH4)), or (ii) a polypeptide wherein the Fab heavy chain of athird Fab molecule shares a carboxy-terminal peptide bond with an Fcdomain subunit (VH₍₃₎-CH1₍₃₎-CH2-CH3(-CH4)) and the Fab light chainpolypeptide of a third Fab molecule (VL₍₃₎-CL₍₃₎). In certainembodiments the polypeptides are covalently linked, e.g., by a disulfidebond.

In some embodiments, the bispecific antigen binding molecule comprises apolypeptide wherein the Fab heavy chain variable region of the secondFab molecule shares a carboxy-terminal peptide bond with the Fab lightchain constant region of the second Fab molecule (i.e. the second Fabmolecule comprises a crossover Fab heavy chain, wherein the heavy chainconstant region is replaced by a light chain constant region), which inturn shares a carboxy-terminal peptide bond with the Fab heavy chain ofthe first Fab molecule, which in turn shares a carboxy-terminal peptidebond with an Fc domain subunit (VH₍₂₎-CL₍₂₎-VH₍₁₎-CH1₍₁₎-CH2-CH3(-CH4)).In other embodiments, the bispecific antigen binding molecule comprisesa polypeptide wherein the Fab heavy chain of the first Fab moleculeshares a carboxy-terminal peptide bond with the Fab heavy chain variableregion of the second Fab molecule which in turn shares acarboxy-terminal peptide bond with the Fab light chain constant regionof the second Fab molecule (i.e. the second Fab molecule comprises acrossover Fab heavy chain, wherein the heavy chain constant region isreplaced by a light chain constant region), which in turn shares acarboxy-terminal peptide bond with an Fc domain subunit(VH₍₁₎-CH1₍₁₎-VH₍₂₎-CL₍₂₎-CH2-CH3(-CH4)).

In some of these embodiments the bispecific antigen binding moleculefurther comprises a crossover Fab light chain polypeptide of the secondFab molecule, wherein the Fab light chain variable region of the secondFab molecule shares a carboxy-terminal peptide bond with the Fab heavychain constant region of the second Fab molecule (VL₍₂₎-CH1₍₂₎), and theFab light chain polypeptide of the first Fab molecule (VL₍₁₎-CL₍₁₎). Inothers of these embodiments the bispecific antigen binding moleculefurther comprises a polypeptide wherein the Fab light chain variableregion of the second Fab molecule shares a carboxy-terminal peptide bondwith the Fab heavy chain constant region of the second Fab moleculewhich in turn shares a carboxy-terminal peptide bond with the Fab lightchain polypeptide of the first Fab molecule (VL₍₂₎-CH1₍₂₎-VL₍₁₎-CL₍₁₎),or a polypeptide wherein the Fab light chain polypeptide of the firstFab molecule shares a carboxy-terminal peptide bond with the Fab heavychain variable region of the second Fab molecule which in turn shares acarboxy-terminal peptide bond with the Fab light chain constant regionof the second Fab molecule (VL₍₁₎-CL₍₁₎-VH₍₂₎-CL₍₂₎), as appropriate.

The bispecific antigen binding molecule according to these embodimentsmay further comprise (i) an Fc domain subunit polypeptide(CH2-CH3(-CH4)), or (ii) a polypeptide wherein the Fab heavy chain of athird Fab molecule shares a carboxy-terminal peptide bond with an Fcdomain subunit (VH₍₃₎-CH1₍₃₎-CH2-CH3(-CH4)) and the Fab light chainpolypeptide of a third Fab molecule (VL₍₃₎-CL₍₃₎). In certainembodiments the polypeptides are covalently linked, e.g., by a disulfidebond.

In certain embodiments, the bispecific antigen binding molecule does notcomprise an Fc domain. In particular such embodiments, said first and,if present third Fab molecules are each a conventional Fab molecule, andthe second Fab molecule is a crossover Fab molecule as described herein,i.e. a Fab molecule wherein the variable domains VH and VL or theconstant domains CL and CH1 of the Fab heavy and light chains areexchanged/replaced by each other. In other such embodiments, said firstand, if present third Fab molecules are each a crossover Fab moleculeand the second Fab molecule is a conventional Fab molecule.

In one such embodiment, the bispecific antigen binding moleculeessentially consists of the first and the second antigen binding moiety,and optionally one or more peptide linkers, wherein the first and thesecond antigen binding moiety are both Fab molecules and the firstantigen binding moiety is fused at the C-terminus of the Fab heavy chainto the N-terminus of the Fab heavy chain of the second antigen bindingmoiety. Such a configuration is schematically depicted in FIGS. 10 and1S (in these examples with the second antigen binding domain being aVH/VL crossover Fab molecule and the first antigen binding moiety beinga conventional Fab molecule).

In another such embodiment, the bispecific antigen binding moleculeessentially consists of the first and the second antigen binding moiety,and optionally one or more peptide linkers, wherein the first and thesecond antigen binding moiety are both Fab molecules and the secondantigen binding moiety is fused at the C-terminus of the Fab heavy chainto the N-terminus of the Fab heavy chain of the first antigen bindingmoiety. Such a configuration is schematically depicted in FIGS. 1P and1T (in these examples with the second antigen binding domain being aVH/VL crossover Fab molecule and the first antigen binding moiety beinga conventional Fab molecule).

In some embodiments, the first Fab molecule is fused at the C-terminusof the Fab heavy chain to the N-terminus of the Fab heavy chain of thesecond Fab molecule, and the bispecific antigen binding molecule furthercomprises a third antigen binding moiety, particularly a third Fabmolecule, wherein said third Fab molecule is fused at the C-terminus ofthe Fab heavy chain to the N-terminus of the Fab heavy chain of thefirst Fab molecule. In certain such embodiments, the bispecific antigenbinding molecule essentially consists of the first, the second and thethird Fab molecule, and optionally one or more peptide linkers, whereinthe first Fab molecule is fused at the C-terminus of the Fab heavy chainto the N-terminus of the Fab heavy chain of the second Fab molecule, andthe third Fab molecule is fused at the C-terminus of the Fab heavy chainto the N-terminus of the Fab heavy chain of the first Fab molecule. Sucha configuration is schematically depicted in FIGS. 1Q and 1U (in theseexamples with the second antigen binding domain being a VH/VL crossoverFab molecule and the first and the antigen binding moiety each being aconventional Fab molecule), or FIGS. 1X and 1Z (in these examples withthe second antigen binding domain being a conventional Fab molecule andthe first and the third antigen binding moiety each being a VH/VLcrossover Fab molecule).

In some embodiments, the second Fab molecule is fused at the C-terminusof the Fab heavy chain to the N-terminus of the Fab heavy chain of thefirst Fab molecule, and the bispecific antigen binding molecule furthercomprises a third antigen binding moiety, particularly a third Fabmolecule, wherein said third Fab molecule is fused at the N-terminus ofthe Fab heavy chain to the C-terminus of the Fab heavy chain of thefirst Fab molecule. In certain such embodiments, the bispecific antigenbinding molecule essentially consists of the first, the second and thethird Fab molecule, and optionally one or more peptide linkers, whereinthe second Fab molecule is fused at the C-terminus of the Fab heavychain to the N-terminus of the Fab heavy chain of the first Fabmolecule, and the third Fab molecule is fused at the N-terminus of theFab heavy chain to the C-terminus of the Fab heavy chain of the firstFab molecule. Such a configuration is schematically depicted in FIGS. 1Rand 1V (in these examples with the second antigen binding domain being aVH/VL crossover Fab molecule and the first and the antigen bindingmoiety each being a conventional Fab molecule), or FIGS. 1W and 1Y (inthese examples with the second antigen binding domain being aconventional Fab molecule and the first and the third antigen bindingmoiety each being a VH/VL crossover Fab molecule).

In certain embodiments the bispecific antigen binding molecule accordingto the invention comprises a polypeptide wherein the Fab heavy chain ofthe first Fab molecule shares a carboxy-terminal peptide bond with theFab light chain variable region of the second Fab molecule, which inturn shares a carboxy-terminal peptide bond with the Fab heavy chainconstant region of the second Fab molecule (i.e. the second Fab moleculecomprises a crossover Fab heavy chain, wherein the heavy chain variableregion is replaced by a light chain variable region)(VH₍₁₎-CH1₍₁₎-VL₍₂₎-CH1₍₂₎). In some embodiments the bispecific antigenbinding molecule further comprises a polypeptide wherein the Fab heavychain variable region of the second Fab molecule shares acarboxy-terminal peptide bond with the Fab light chain constant regionof the second Fab molecule (VH₍₂₎-CL₍₂₎) and the Fab light chainpolypeptide of the first Fab molecule (VL₍₁₎-CL₍₁₎). In certainembodiments the bispecific antigen binding molecule according to theinvention comprises a polypeptide wherein the Fab light chain variableregion of the second Fab molecule shares a carboxy-terminal peptide bondwith the Fab heavy chain constant region of the second Fab molecule(i.e. the second Fab molecule comprises a crossover Fab heavy chain,wherein the heavy chain variable region is replaced by a light chainvariable region), which in turn shares a carboxy-terminal peptide bondwith the Fab heavy chain of the first Fab molecule(VL₍₂₎-CH1₍₂₎-VH₍₁₎-CH1₍₂₎). In some embodiments the bispecific antigenbinding molecule further comprises a polypeptide wherein the Fab heavychain variable region of the second Fab molecule shares acarboxy-terminal peptide bond with the Fab light chain constant regionof the second Fab molecule (VH₍₂₎-CL₍₂₎) and the Fab light chainpolypeptide of the first Fab molecule (VL₍₁₎-CL₍₁₎). In certainembodiments the bispecific antigen binding molecule according to theinvention comprises a polypeptide wherein the Fab heavy chain variableregion of the second Fab molecule shares a carboxy-terminal peptide bondwith the Fab light chain constant region of the second Fab molecule(i.e. the second Fab molecule comprises a crossover Fab heavy chain,wherein the heavy chain constant region is replaced by a light chainconstant region), which in turn shares a carboxy-terminal peptide bondwith the Fab heavy chain of the first Fab molecule(VH₍₂₎-CL₍₂₎-VH₍₁₎-CH1₍₁₎). In some embodiments the bispecific antigenbinding molecule further comprises a polypeptide wherein the Fab lightchain variable region of the second Fab molecule shares acarboxy-terminal peptide bond with the Fab heavy chain constant regionof the second Fab molecule (VL₍₂₎-CH1₍₂₎) and the Fab light chainpolypeptide of the first Fab molecule (VL₍₁₎-CL₍₁₎). In certainembodiments the bispecific antigen binding molecule according to theinvention comprises a polypeptide wherein the Fab light chain variableregion of the second Fab molecule shares a carboxy-terminal peptide bondwith the Fab heavy chain constant region of the second Fab molecule(i.e. the second Fab molecule comprises a crossover Fab heavy chain,wherein the heavy chain variable region is replaced by a light chainvariable region), which in turn shares a carboxy-terminal peptide bondwith the Fab heavy chain of the first Fab molecule(VL₍₂₎-CH1₍₂₎-VH₍₁₎-CH1₍₁₎). In some embodiments the bispecific antigenbinding molecule further comprises a polypeptide wherein the Fab heavychain variable region of the second Fab molecule shares acarboxy-terminal peptide bond with the Fab light chain constant regionof the second Fab molecule (VH₍₂₎-CL₍₂₎) and the Fab light chainpolypeptide of the first Fab molecule (VL₍₁₎-CL₍₁₎).

In certain embodiments the bispecific antigen binding molecule accordingto the invention comprises a polypeptide wherein the Fab heavy chain ofa third Fab molecule shares a carboxy-terminal peptide bond with the Fabheavy chain of the first Fab molecule, which in turn shares acarboxy-terminal peptide bond with the Fab light chain variable regionof the second Fab molecule, which in turn shares a carboxy-terminalpeptide bond with the Fab heavy chain constant region of the second Fabmolecule (i.e. the second Fab molecule comprises a crossover Fab heavychain, wherein the heavy chain variable region is replaced by a lightchain variable region) (VH₍₃₎-CH1₍₃₎-VH₍₁₎-CH1₍₁₎-VL₍₂₎-CH1₍₂₎). In someembodiments the bispecific antigen binding molecule further comprises apolypeptide wherein the Fab heavy chain variable region of the secondFab molecule shares a carboxy-terminal peptide bond with the Fab lightchain constant region of the second Fab molecule (VH₍₂₎-CL₍₂₎) and theFab light chain polypeptide of the first Fab molecule (VL₍₁₎-CL₍₁₎). Insome embodiments the bispecific antigen binding molecule furthercomprises the Fab light chain polypeptide of a third Fab molecule(VL₍₃₎-CL₍₃₎).

In certain embodiments the bispecific antigen binding molecule accordingto the invention comprises a polypeptide wherein the Fab heavy chain ofa third Fab molecule shares a carboxy-terminal peptide bond with the Fabheavy chain of the first Fab molecule, which in turn shares acarboxy-terminal peptide bond with the Fab heavy chain variable regionof the second Fab molecule, which in turn shares a carboxy-terminalpeptide bond with the Fab light chain constant region of the second Fabmolecule (i.e. the second Fab molecule comprises a crossover Fab heavychain, wherein the heavy chain constant region is replaced by a lightchain constant region) (VH₍₃₎-CH1₍₃₎-VH₍₁₎-CH1₍₁₎-VH₍₂₎-CL₍₂₎). In someembodiments the bispecific antigen binding molecule further comprises apolypeptide wherein the Fab light chain variable region of the secondFab molecule shares a carboxy-terminal peptide bond with the Fab heavychain constant region of the second Fab molecule (VL₍₂₎-CH1₍₂₎) and theFab light chain polypeptide of the first Fab molecule (VL₍₁₎-CL₍₁₎). Insome embodiments the bispecific antigen binding molecule furthercomprises the Fab light chain polypeptide of a third Fab molecule(VL₍₃₎-CL₍₃₎).

In certain embodiments the bispecific antigen binding molecule accordingto the invention comprises a polypeptide wherein the Fab light chainvariable region of the second Fab molecule shares a carboxy-terminalpeptide bond with the Fab heavy chain constant region of the second Fabmolecule (i.e. the second Fab molecule comprises a crossover Fab heavychain, wherein the heavy chain variable region is replaced by a lightchain variable region), which in turn shares a carboxy-terminal peptidebond with the Fab heavy chain of the first Fab molecule, which in turnshares a carboxy-terminal peptide bond with the Fab heavy chain of athird Fab molecule (VL₍₂₎-CH1₍₂₎-VH₍₁₎-CH1₍₁₎-VH₍₃₎-CH1₍₃₎). In someembodiments the bispecific antigen binding molecule further comprises apolypeptide wherein the Fab heavy chain variable region of the secondFab molecule shares a carboxy-terminal peptide bond with the Fab lightchain constant region of the second Fab molecule (VH₍₂₎-CL₍₂₎) and theFab light chain polypeptide of the first Fab molecule (VL₍₁₎-CL₍₁₎). Insome embodiments the bispecific antigen binding molecule furthercomprises the Fab light chain polypeptide of a third Fab molecule(VL₍₃₎-CL₍₃₎).

In certain embodiments the bispecific antigen binding molecule accordingto the invention comprises a polypeptide wherein the Fab heavy chainvariable region of the second Fab molecule shares a carboxy-terminalpeptide bond with the Fab light chain constant region of the second Fabmolecule (i.e. the second Fab molecule comprises a crossover Fab heavychain, wherein the heavy chain constant region is replaced by a lightchain constant region), which in turn shares a carboxy-terminal peptidebond with the Fab heavy chain of the first Fab molecule, which in turnshares a carboxy-terminal peptide bond with the Fab heavy chain of athird Fab molecule (VH₍₂₎-CL₍₂₎-VH₍₁₎-CH1₍₁₎-VH₍₃₎-CH1₍₃₎). In someembodiments the bispecific antigen binding molecule further comprises apolypeptide wherein the Fab light chain variable region of the secondFab molecule shares a carboxy-terminal peptide bond with the Fab heavychain constant region of the second Fab molecule (VL₍₂₎-CH1₍₂₎) and theFab light chain polypeptide of the first Fab molecule (VL₍₁₎-CL₍₁₎). Insome embodiments the bispecific antigen binding molecule furthercomprises the Fab light chain polypeptide of a third Fab molecule(VL₍₃₎-CL₍₃₎).

In certain embodiments the bispecific antigen binding molecule accordingto the invention comprises a polypeptide wherein the Fab heavy chain ofthe second Fab molecule shares a carboxy-terminal peptide bond with theFab light chain variable region of the first Fab molecule, which in turnshares a carboxy-terminal peptide bond with the Fab heavy chain constantregion of the first Fab molecule (i.e. the first Fab molecule comprisesa crossover Fab heavy chain, wherein the heavy chain variable region isreplaced by a light chain variable region), which in turn shares acarboxy-terminal peptide bond with the Fab light chain variable regionof a third Fab molecule, which in turn shares a carboxy-terminal peptidebond with the Fab heavy chain constant region of a third Fab molecule(i.e. the third Fab molecule comprises a crossover Fab heavy chain,wherein the heavy chain variable region is replaced by a light chainvariable region) (VH₍₂₎-CH1₍₂₎-VL₍₁₎-CH1₍₁₎-VL₍₃₎-CH1₍₃₎). In someembodiments the bispecific antigen binding molecule further comprises apolypeptide wherein the Fab heavy chain variable region of the first Fabmolecule shares a carboxy-terminal peptide bond with the Fab light chainconstant region of the first Fab molecule (VH₍₁₎-CL₍₁₎) and the Fablight chain polypeptide of the second Fab molecule (VL₍₂₎-CL₍₂₎). Insome embodiments the bispecific antigen binding molecule furthercomprises a polypeptide wherein the Fab heavy chain variable region of athird Fab molecule shares a carboxy-terminal peptide bond with the Fablight chain constant region of a third Fab molecule (VH₍₃₎-CL₍₃₎).

In certain embodiments the bispecific antigen binding molecule accordingto the invention comprises a polypeptide wherein the Fab heavy chain ofthe second Fab molecule shares a carboxy-terminal peptide bond with theFab heavy chain variable region of the first Fab molecule, which in turnshares a carboxy-terminal peptide bond with the Fab light chain constantregion of the first Fab molecule (i.e. the first Fab molecule comprisesa crossover Fab heavy chain, wherein the heavy chain constant region isreplaced by a light chain constant region), which in turn shares acarboxy-terminal peptide bond with the Fab heavy chain variable regionof a third Fab molecule, which in turn shares a carboxy-terminal peptidebond with the Fab light chain constant region of a third Fab molecule(i.e. the third Fab molecule comprises a crossover Fab heavy chain,wherein the heavy chain constant region is replaced by a light chainconstant region) (VH₍₂₎-CH1₍₂₎-VH₍₁₎-CL₍₁₎-VH₍₃₎-CL₍₃₎). In someembodiments the bispecific antigen binding molecule further comprises apolypeptide wherein the Fab light chain variable region of the first Fabmolecule shares a carboxy-terminal peptide bond with the Fab heavy chainconstant region of the first Fab molecule (VL₍₁₎-CH1₍₁₎) and the Fablight chain polypeptide of the second Fab molecule (VL₍₂₎-CL₍₂₎). Insome embodiments the bispecific antigen binding molecule furthercomprises a polypeptide wherein the Fab light chain variable region of athird Fab molecule shares a carboxy-terminal peptide bond with the Fabheavy chain constant region of a third Fab molecule (VL₍₃₎-CH1₍₃₎).

In certain embodiments the bispecific antigen binding molecule accordingto the invention comprises a polypeptide wherein the Fab light chainvariable region of a third Fab molecule shares a carboxy-terminalpeptide bond with the Fab heavy chain constant region of a third Fabmolecule (i.e. the third Fab molecule comprises a crossover Fab heavychain, wherein the heavy chain variable region is replaced by a lightchain variable region), which in turn shares a carboxy-terminal peptidebond with the Fab light chain variable region of the first Fab molecule,which in turn shares a carboxy-terminal peptide bond with the Fab heavychain constant region of the first Fab molecule (i.e. the first Fabmolecule comprises a crossover Fab heavy chain, wherein the heavy chainvariable region is replaced by a light chain variable region), which inturn shares a carboxy-terminal peptide bond with the Fab heavy chain ofthe second Fab molecule (VL₍₃₎-CH1₍₃₎-VL₍₁₎-CH1₍₁₎-VH₍₂₎-CH1₍₂₎). Insome embodiments the bispecific antigen binding molecule furthercomprises a polypeptide wherein the Fab heavy chain variable region ofthe first Fab molecule shares a carboxy-terminal peptide bond with theFab light chain constant region of the first Fab molecule (VH₍₁₎-CL₍₁₎)and the Fab light chain polypeptide of the second Fab molecule(VL₍₂₎-CL₍₂₎). In some embodiments the bispecific antigen bindingmolecule further comprises a polypeptide wherein the Fab heavy chainvariable region of a third Fab molecule shares a carboxy-terminalpeptide bond with the Fab light chain constant region of a third Fabmolecule (VH₍₃₎-CL₍₃₎).

In certain embodiments the bispecific antigen binding molecule accordingto the invention comprises a polypeptide wherein the Fab heavy chainvariable region of a third Fab molecule shares a carboxy-terminalpeptide bond with the Fab light chain constant region of a third Fabmolecule (i.e. the third Fab molecule comprises a crossover Fab heavychain, wherein the heavy chain constant region is replaced by a lightchain constant region), which in turn shares a carboxy-terminal peptidebond with the Fab heavy chain variable region of the first Fab molecule,which in turn shares a carboxy-terminal peptide bond with the Fab lightchain constant region of the first Fab molecule (i.e. the first Fabmolecule comprises a crossover Fab heavy chain, wherein the heavy chainconstant region is replaced by a light chain constant region), which inturn shares a carboxy-terminal peptide bond with the Fab heavy chain ofthe second Fab molecule (VH₍₃₎-CL₍₃₎-VH₍₁₎-CL₍₁₎-VH₍₂₎-CH1₍₂₎). In someembodiments the bispecific antigen binding molecule further comprises apolypeptide wherein the Fab light chain variable region of the first Fabmolecule shares a carboxy-terminal peptide bond with the Fab heavy chainconstant region of the first Fab molecule (VL₍₁₎-CH1₍₁₎) and the Fablight chain polypeptide of the second Fab molecule (VL₍₂₎-CL₍₂₎). Insome embodiments the bispecific antigen binding molecule furthercomprises a polypeptide wherein the Fab light chain variable region of athird Fab molecule shares a carboxy-terminal peptide bond with the Fabheavy chain constant region of a third Fab molecule (VL₍₃₎-CH1₍₃₎).

In one embodiment, the invention provides a bispecific antigen bindingmolecule comprising

a) a first antigen binding moiety that binds to a first antigen, whereinthe first antigen is HLA-A2/WT1, particularly HLA-A2/WT1_(RMF), and thefirst antigen binding moiety is a Fab molecule comprising a heavy chainvariable region (VH) comprising a heavy chain complementary determiningregion (HCDR) 1 of SEQ ID NO: 1, a HCDR 2 of SEQ ID NO: 2, and a HCDR 3of SEQ ID NO: 3, and a light chain variable region (VL) comprising alight chain complementarity determining region (LCDR) 1 of SEQ ID NO: 4,a LCDR 2 of SEQ ID NO: 5 and a LCDR 3 of SEQ ID NO: 6;b) a second antigen binding moiety that binds to a second antigen,wherein the second antigen is an activating T cell antigen, particularlyCD3, more particularly CD3 epsilon, and the second antigen bindingmoiety is a Fab molecule wherein the variable domains VL and VH or theconstant domains CL and CH1 of the Fab light chain and the Fab heavychain are replaced by each other;c) an Fc domain composed of a first and a second subunit;wherein(i) the first antigen binding moiety under a) is fused at the C-terminusof the Fab heavy chain to the N-terminus of the Fab heavy chain of thesecond antigen binding moiety under b), and the second antigen bindingmoiety under b) is fused at the C-terminus of the Fab heavy chain to theN-terminus of one of the subunits of the Fc domain under c), or(ii) the second antigen binding moiety under b) is fused at theC-terminus of the Fab heavy chain to the N-terminus of the Fab heavychain of the first antigen binding moiety under a), and the firstantigen binding moiety under a) is fused at the C-terminus of the Fabheavy chain to the N-terminus of one of the subunits of the Fc domainunder c).

In a particular embodiment, the invention provides a bispecific antigenbinding molecule comprising

a) a first antigen binding moiety that binds to a first antigen, whereinthe first antigen is HLA-A2/WT1, particularly HLA-A2/WT1_(RMF), and thefirst antigen binding moiety is a Fab molecule comprising a heavy chainvariable region (VH) comprising a heavy chain complementary determiningregion (HCDR) 1 of SEQ ID NO: 1, a HCDR 2 of SEQ ID NO: 2, and a HCDR 3of SEQ ID NO: 3, and a light chain variable region (VL) comprising alight chain complementarity determining region (LCDR) 1 of SEQ ID NO: 4,a LCDR 2 of SEQ ID NO: 5 and a LCDR 3 of SEQ ID NO: 6;b) a second antigen binding moiety that binds to a second antigen,wherein the second antigen is an activating T cell antigen, particularlyCD3, more particularly CD3 epsilon, and the second antigen bindingmoiety is a Fab molecule wherein the variable domains VL and VH or theconstant domains CL and CH1 of the Fab light chain and the Fab heavychain are replaced by each other;c) a third antigen binding moiety that binds to the first antigen and isidentical to the first antigen binding moiety; andd) an Fc domain composed of a first and a second subunit;wherein(i) the first antigen binding moiety under a) is fused at the C-terminusof the Fab heavy chain to the N-terminus of the Fab heavy chain of thesecond antigen binding moiety under b), and the second antigen bindingmoiety under b) and the third antigen binding moiety under c) are eachfused at the C-terminus of the Fab heavy chain to the N-terminus of oneof the subunits of the Fc domain under d), or(ii) the second antigen binding moiety under b) is fused at theC-terminus of the Fab heavy chain to the N-terminus of the Fab heavychain of the first antigen binding moiety under a), and the firstantigen binding moiety under a) and the third antigen binding moietyunder c) are each fused at the C-terminus of the Fab heavy chain to theN-terminus of one of the subunits of the Fc domain under d).

In another embodiment, the invention provides a bispecific antigenbinding molecule comprising

a) a first antigen binding moiety that binds to a first antigen, whereinthe first antigen is HLA-A2/WT1, particularly HLA-A2/WT1_(RMF), and thefirst antigen binding moiety is a Fab molecule comprising a heavy chainvariable region (VH) comprising a heavy chain complementary determiningregion (HCDR) 1 of SEQ ID NO: 1, a HCDR 2 of SEQ ID NO: 2, and a HCDR 3of SEQ ID NO: 3, and a light chain variable region (VL) comprising alight chain complementarity determining region (LCDR) 1 of SEQ ID NO: 4,a LCDR 2 of SEQ ID NO: 5 and a LCDR 3 of SEQ ID NO: 6;b) a second antigen binding moiety that binds to a second antigen,wherein the second antigen is an activating T cell antigen, particularlyCD3, more particularly CD3 epsilon, and the second antigen bindingmoiety is a Fab molecule wherein the variable domains VL and VH or theconstant domains CL and CH1 of the Fab light chain and the Fab heavychain are replaced by each other;c) an Fc domain composed of a first and a second subunit;wherein(i) the first antigen binding moiety under a) and the second antigenbinding moiety under b) are each fused at the C-terminus of the Fabheavy chain to the N-terminus of one of the subunits of the Fc domainunder c).

In one embodiment, the invention provides a bispecific antigen bindingmolecule comprising

a) a first antigen binding moiety that binds to a first antigen, whereinthe first antigen is HLA-A2/WT1, particularly HLA-A2/WT1_(RMF), and thefirst antigen binding moiety is a Fab molecule comprising a heavy chainvariable region (VH) comprising a heavy chain complementary determiningregion (HCDR) 1 of SEQ ID NO: 9, a HCDR 2 of SEQ ID NO: 10, and a HCDR 3of SEQ ID NO: 11, and a light chain variable region (VL) comprising alight chain complementarity determining region (LCDR) 1 of SEQ ID NO:12, a LCDR 2 of SEQ ID NO: 13 and a LCDR 3 of SEQ ID NO: 14;b) a second antigen binding moiety that binds to a second antigen,wherein the second antigen is an activating T cell antigen, particularlyCD3, more particularly CD3 epsilon, and the second antigen bindingmoiety is a Fab molecule wherein the variable domains VL and VH or theconstant domains CL and CH1 of the Fab light chain and the Fab heavychain are replaced by each other;c) an Fc domain composed of a first and a second subunit;wherein(i) the first antigen binding moiety under a) is fused at the C-terminusof the Fab heavy chain to the N-terminus of the Fab heavy chain of thesecond antigen binding moiety under b), and the second antigen bindingmoiety under b) is fused at the C-terminus of the Fab heavy chain to theN-terminus of one of the subunits of the Fc domain under c), or(ii) the second antigen binding moiety under b) is fused at theC-terminus of the Fab heavy chain to the N-terminus of the Fab heavychain of the first antigen binding moiety under a), and the firstantigen binding moiety under a) is fused at the C-terminus of the Fabheavy chain to the N-terminus of one of the subunits of the Fc domainunder c).

In a particular embodiment, the invention provides a bispecific antigenbinding molecule comprising

a) a first antigen binding moiety that binds to a first antigen, whereinthe first antigen is HLA-A2/WT1, particularly HLA-A2/WT1_(RMF), and thefirst antigen binding moiety is a Fab molecule comprising a heavy chainvariable region (VH) comprising a heavy chain complementary determiningregion (HCDR) 1 of SEQ ID NO: 9, a HCDR 2 of SEQ ID NO: 10, and a HCDR 3of SEQ ID NO: 11, and a light chain variable region (VL) comprising alight chain complementarity determining region (LCDR) 1 of SEQ ID NO:12, a LCDR 2 of SEQ ID NO: 13 and a LCDR 3 of SEQ ID NO: 14;b) a second antigen binding moiety that binds to a second antigen,wherein the second antigen is an activating T cell antigen, particularlyCD3, more particularly CD3 epsilon, and the second antigen bindingmoiety is a Fab molecule wherein the variable domains VL and VH or theconstant domains CL and CH1 of the Fab light chain and the Fab heavychain are replaced by each other;c) a third antigen binding moiety that binds to the first antigen and isidentical to the first antigen binding moiety; andd) an Fc domain composed of a first and a second subunit;wherein(i) the first antigen binding moiety under a) is fused at the C-terminusof the Fab heavy chain to the N-terminus of the Fab heavy chain of thesecond antigen binding moiety under b), and the second antigen bindingmoiety under b) and the third antigen binding moiety under c) are eachfused at the C-terminus of the Fab heavy chain to the N-terminus of oneof the subunits of the Fc domain under d), or(ii) the second antigen binding moiety under b) is fused at theC-terminus of the Fab heavy chain to the N-terminus of the Fab heavychain of the first antigen binding moiety under a), and the firstantigen binding moiety under a) and the third antigen binding moietyunder c) are each fused at the C-terminus of the Fab heavy chain to theN-terminus of one of the subunits of the Fc domain under d).

In another embodiment, the invention provides a bispecific antigenbinding molecule comprising

a) a first antigen binding moiety that binds to a first antigen, whereinthe first antigen is HLA-A2/WT1, particularly HLA-A2/WT1_(RMF), and thefirst antigen binding moiety is a Fab molecule comprising a heavy chainvariable region (VH) comprising a heavy chain complementary determiningregion (HCDR) 1 of SEQ ID NO: 9, a HCDR 2 of SEQ ID NO: 10, and a HCDR 3of SEQ ID NO: 11, and a light chain variable region (VL) comprising alight chain complementarity determining region (LCDR) 1 of SEQ ID NO:12, a LCDR 2 of SEQ ID NO: 13 and a LCDR 3 of SEQ ID NO: 14;b) a second antigen binding moiety that binds to a second antigen,wherein the second antigen is an activating T cell antigen, particularlyCD3, more particularly CD3 epsilon, and the second antigen bindingmoiety is a Fab molecule wherein the variable domains VL and VH or theconstant domains CL and CH1 of the Fab light chain and the Fab heavychain are replaced by each other;c) an Fc domain composed of a first and a second subunit;wherein(i) the first antigen binding moiety under a) and the second antigenbinding moiety under b) are each fused at the C-terminus of the Fabheavy chain to the N-terminus of one of the subunits of the Fc domainunder c).

In all of the different configurations of the bispecific antigen bindingmolecule according to the invention, the amino acid substitutionsdescribed herein, if present, may either be in the CH1 and CL domains ofthe first and (if present) the third antigen binding moiety/Fabmolecule, or in the CH1 and CL domains of the second antigen bindingmoiety/Fab molecule. Preferably, they are in the CH1 and CL domains ofthe first and (if present) the third antigen binding moiety/Fabmolecule. In accordance with the concept of the invention, if amino acidsubstitutions as described herein are made in the first (and, ifpresent, the third) antigen binding moiety/Fab molecule, no such aminoacid substitutions are made in the second antigen binding moiety/Fabmolecule. Conversely, if amino acid substitutions as described hereinare made in the second antigen binding moiety/Fab molecule, no suchamino acid substitutions are made in the first (and, if present, thethird) antigen binding moiety/Fab molecule. Amino acid substitutions areparticularly made in bispecific antigen binding molecules comprising aFab molecule wherein the variable domains VL and VH1 of the Fab lightchain and the Fab heavy chain are replaced by each other.

In particular embodiments of the bispecific antigen binding moleculeaccording to the invention, particularly wherein amino acidsubstitutions as described herein are made in the first (and, ifpresent, the third) antigen binding moiety/Fab molecule, the constantdomain CL of the first (and, if present, the third) Fab molecule is ofkappa isotype. In other embodiments of the bispecific antigen bindingmolecule according to the invention, particularly wherein amino acidsubstitutions as described herein are made in the second antigen bindingmoiety/Fab molecule, the constant domain CL of the second antigenbinding moiety/Fab molecule is of kappa isotype. In some embodiments,the constant domain CL of the first (and, if present, the third) antigenbinding moiety/Fab molecule and the constant domain CL of the secondantigen binding moiety/Fab molecule are of kappa isotype.

In one embodiment, the invention provides a bispecific antigen bindingmolecule comprising

a) a first antigen binding moiety that binds to a first antigen, whereinthe first antigen is HLA-A2/WT1, particularly HLA-A2/WT1_(RMF), and thefirst antigen binding moiety is a Fab molecule comprising a heavy chainvariable region (VH) comprising a heavy chain complementary determiningregion (HCDR) 1 of SEQ ID NO: 1, a HCDR 2 of SEQ ID NO: 2, and a HCDR 3of SEQ ID NO: 3, and a light chain variable region (VL) comprising alight chain complementarity determining region (LCDR) 1 of SEQ ID NO: 4,a LCDR 2 of SEQ ID NO: 5 and a LCDR 3 of SEQ ID NO: 6;b) a second antigen binding moiety that binds to a second antigen,wherein the second antigen is an activating T cell antigen, particularlyCD3, more particularly CD3 epsilon, and the second antigen bindingmoiety is a Fab molecule wherein the variable domains VL and VH of theFab light chain and the Fab heavy chain are replaced by each other;c) an Fc domain composed of a first and a second subunit;wherein in the constant domain CL of the first antigen binding moietyunder a) the amino acid at position 124 is substituted by lysine (K)(numbering according to Kabat) and the amino acid at position 123 issubstituted by lysine (K) or arginine (R) (numbering according to Kabat)(most particularly by arginine (R)), and wherein in the constant domainCH1 of the first antigen binding moiety under a) the amino acid atposition 147 is substituted by glutamic acid (E) (numbering according toKabat EU index) and the amino acid at position 213 is substituted byglutamic acid (E) (numbering according to Kabat EU index); andwherein(i) the first antigen binding moiety under a) is fused at the C-terminusof the Fab heavy chain to the N-terminus of the Fab heavy chain of thesecond antigen binding moiety under b), and the second antigen bindingmoiety under b) is fused at the C-terminus of the Fab heavy chain to theN-terminus of one of the subunits of the Fc domain under c), or(ii) the second antigen binding moiety under b) is fused at theC-terminus of the Fab heavy chain to the N-terminus of the Fab heavychain of the first antigen binding moiety under a), and the firstantigen binding moiety under a) is fused at the C-terminus of the Fabheavy chain to the N-terminus of one of the subunits of the Fc domainunder c).

In a particular embodiment, the invention provides a bispecific antigenbinding molecule comprising

a) a first antigen binding moiety that binds to a first antigen, whereinthe first antigen is HLA-A2/WT1, particularly HLA-A2/WT1_(RMF), and thefirst antigen binding moiety is a Fab molecule comprising a heavy chainvariable region (VH) comprising a heavy chain complementary determiningregion (HCDR) 1 of SEQ ID NO: 1, a HCDR 2 of SEQ ID NO: 2, and a HCDR 3of SEQ ID NO: 3, and a light chain variable region (VL) comprising alight chain complementarity determining region (LCDR) 1 of SEQ ID NO: 4,a LCDR 2 of SEQ ID NO: 5 and a LCDR 3 of SEQ ID NO: 6;b) a second antigen binding moiety that binds to a second antigen,wherein the second antigen is an activating T cell antigen, particularlyCD3, more particularly CD3 epsilon, and the second antigen bindingmoiety is a Fab molecule wherein the variable domains VL and VH of theFab light chain and the Fab heavy chain are replaced by each other;c) a third antigen binding moiety that binds to the first antigen and isidentical to the first antigen binding moiety; andd) an Fc domain composed of a first and a second subunit;wherein in the constant domain CL of the first antigen binding moietyunder a) and the third antigen binding moiety under c) the amino acid atposition 124 is substituted by lysine (K) (numbering according to Kabat)and the amino acid at position 123 is substituted by lysine (K) orarginine (R) (numbering according to Kabat) (most particularly byarginine (R)), and wherein in the constant domain CH1 of the firstantigen binding moiety under a) and the third antigen binding moietyunder c) the amino acid at position 147 is substituted by glutamic acid(E) (numbering according to Kabat EU index) and the amino acid atposition 213 is substituted by glutamic acid (E) (numbering according toKabat EU index); andwherein(i) the first antigen binding moiety under a) is fused at the C-terminusof the Fab heavy chain to the N-terminus of the Fab heavy chain of thesecond antigen binding moiety under b), and the second antigen bindingmoiety under b) and the third antigen binding moiety under c) are eachfused at the C-terminus of the Fab heavy chain to the N-terminus of oneof the subunits of the Fc domain under d), or(ii) the second antigen binding moiety under b) is fused at theC-terminus of the Fab heavy chain to the N-terminus of the Fab heavychain of the first antigen binding moiety under a), and the firstantigen binding moiety under a) and the third antigen binding moietyunder c) are each fused at the C-terminus of the Fab heavy chain to theN-terminus of one of the subunits of the Fc domain under d).

In another embodiment, the invention provides a bispecific antigenbinding molecule comprising

a) a first antigen binding moiety that binds to a first antigen, whereinthe first antigen is HLA-A2/WT1, particularly HLA-A2/WT1_(RMF), and thefirst antigen binding moiety is a Fab molecule comprising a heavy chainvariable region (VH) comprising a heavy chain complementary determiningregion (HCDR) 1 of SEQ ID NO: 1, a HCDR 2 of SEQ ID NO: 2, and a HCDR 3of SEQ ID NO: 3, and a light chain variable region (VL) comprising alight chain complementarity determining region (LCDR) 1 of SEQ ID NO: 4,a LCDR 2 of SEQ ID NO: 5 and a LCDR 3 of SEQ ID NO: 6;b) a second antigen binding moiety that binds to a second antigen,wherein the second antigen is an activating T cell antigen, particularlyCD3, more particularly CD3 epsilon, and the second antigen bindingmoiety is a Fab molecule wherein the variable domains VL and VH of theFab light chain and the Fab heavy chain are replaced by each other;c) an Fc domain composed of a first and a second subunit;wherein in the constant domain CL of the first antigen binding moietyunder a) the amino acid at position 124 is substituted by lysine (K)(numbering according to Kabat) and the amino acid at position 123 issubstituted by lysine (K) or arginine (R) (numbering according to Kabat)(most particularly by arginine (R)), and wherein in the constant domainCH1 of the first antigen binding moiety under a) the amino acid atposition 147 is substituted by glutamic acid (E) (numbering according toKabat EU index) and the amino acid at position 213 is substituted byglutamic acid (E) (numbering according to Kabat EU index); andwherein the first antigen binding moiety under a) and the second antigenbinding moiety under b) are each fused at the C-terminus of the Fabheavy chain to the N-terminus of one of the subunits of the Fc domainunder c).

In one embodiment, the invention provides a bispecific antigen bindingmolecule comprising a) a first antigen binding moiety that binds to afirst antigen, wherein the first antigen is HLA-A2/WT1, particularlyHLA-A2/WT1_(RMF), and the first antigen binding moiety is a Fab moleculecomprising a heavy chain variable region (VH) comprising a heavy chaincomplementary determining region (HCDR) 1 of SEQ ID NO: 9, a HCDR 2 ofSEQ ID NO: 10, and a HCDR 3 of SEQ ID NO: 11, and a light chain variableregion (VL) comprising a light chain complementarity determining region(LCDR) 1 of SEQ ID NO: 12, a LCDR 2 of SEQ ID NO: 13 and a LCDR 3 of SEQID NO: 14;

b) a second antigen binding moiety that binds to a second antigen,wherein the second antigen is an activating T cell antigen, particularlyCD3, more particularly CD3 epsilon, and the second antigen bindingmoiety is a Fab molecule wherein the variable domains VL and VH of theFab light chain and the Fab heavy chain are replaced by each other;c) an Fc domain composed of a first and a second subunit;wherein in the constant domain CL of the first antigen binding moietyunder a) the amino acid at position 124 is substituted by lysine (K)(numbering according to Kabat) and the amino acid at position 123 issubstituted by lysine (K) or arginine (R) (numbering according to Kabat)(most particularly by arginine (R)), and wherein in the constant domainCH1 of the first antigen binding moiety under a) the amino acid atposition 147 is substituted by glutamic acid (E) (numbering according toKabat EU index) and the amino acid at position 213 is substituted byglutamic acid (E) (numbering according to Kabat EU index); andwherein(i) the first antigen binding moiety under a) is fused at the C-terminusof the Fab heavy chain to the N-terminus of the Fab heavy chain of thesecond antigen binding moiety under b), and the second antigen bindingmoiety under b) is fused at the C-terminus of the Fab heavy chain to theN-terminus of one of the subunits of the Fc domain under c), or(ii) the second antigen binding moiety under b) is fused at theC-terminus of the Fab heavy chain to the N-terminus of the Fab heavychain of the first antigen binding moiety under a), and the firstantigen binding moiety under a) is fused at the C-terminus of the Fabheavy chain to the N-terminus of one of the subunits of the Fc domainunder c).

In a particular embodiment, the invention provides a bispecific antigenbinding molecule comprising

a) a first antigen binding moiety that binds to a first antigen, whereinthe first antigen is HLA-A2/WT1, particularly HLA-A2/WT1_(RMF), and thefirst antigen binding moiety is a Fab molecule comprising a heavy chainvariable region (VH) comprising a heavy chain complementary determiningregion (HCDR) 1 of SEQ ID NO: 9, a HCDR 2 of SEQ ID NO: 10, and a HCDR 3of SEQ ID NO: 11, and a light chain variable region (VL) comprising alight chain complementarity determining region (LCDR) 1 of SEQ ID NO:12, a LCDR 2 of SEQ ID NO: 13 and a LCDR 3 of SEQ ID NO: 14;b) a second antigen binding moiety that binds to a second antigen,wherein the second antigen is an activating T cell antigen, particularlyCD3, more particularly CD3 epsilon, and the second antigen bindingmoiety is a Fab molecule wherein the variable domains VL and VH of theFab light chain and the Fab heavy chain are replaced by each other;c) a third antigen binding moiety that binds to the first antigen and isidentical to the first antigen binding moiety; andd) an Fc domain composed of a first and a second subunit;wherein in the constant domain CL of the first antigen binding moietyunder a) and the third antigen binding moiety under c) the amino acid atposition 124 is substituted by lysine (K) (numbering according to Kabat)and the amino acid at position 123 is substituted by lysine (K) orarginine (R) (numbering according to Kabat) (most particularly byarginine (R)), and wherein in the constant domain CH1 of the firstantigen binding moiety under a) and the third antigen binding moietyunder c) the amino acid at position 147 is substituted by glutamic acid(E) (numbering according to Kabat EU index) and the amino acid atposition 213 is substituted by glutamic acid (E) (numbering according toKabat EU index); andwherein(i) the first antigen binding moiety under a) is fused at the C-terminusof the Fab heavy chain to the N-terminus of the Fab heavy chain of thesecond antigen binding moiety under b), and the second antigen bindingmoiety under b) and the third antigen binding moiety under c) are eachfused at the C-terminus of the Fab heavy chain to the N-terminus of oneof the subunits of the Fc domain under d), or(ii) the second antigen binding moiety under b) is fused at theC-terminus of the Fab heavy chain to the N-terminus of the Fab heavychain of the first antigen binding moiety under a), and the firstantigen binding moiety under a) and the third antigen binding moietyunder c) are each fused at the C-terminus of the Fab heavy chain to theN-terminus of one of the subunits of the Fc domain under d).

In another embodiment, the invention provides a bispecific antigenbinding molecule comprising

a) a first antigen binding moiety that binds to a first antigen, whereinthe first antigen is HLA-A2/WT1, particularly HLA-A2/WT1_(RMF), and thefirst antigen binding moiety is a Fab molecule comprising a heavy chainvariable region (VH) comprising a heavy chain complementary determiningregion (HCDR) 1 of SEQ ID NO: 9, a HCDR 2 of SEQ ID NO: 10, and a HCDR 3of SEQ ID NO: 11, and a light chain variable region (VL) comprising alight chain complementarity determining region (LCDR) 1 of SEQ ID NO:12, a LCDR 2 of SEQ ID NO: 13 and a LCDR 3 of SEQ ID NO: 14;b) a second antigen binding moiety that binds to a second antigen,wherein the second antigen is an activating T cell antigen, particularlyCD3, more particularly CD3 epsilon, and the second antigen bindingmoiety is a Fab molecule wherein the variable domains VL and VH of theFab light chain and the Fab heavy chain are replaced by each other;c) an Fc domain composed of a first and a second subunit;wherein in the constant domain CL of the first antigen binding moietyunder a) the amino acid at position 124 is substituted by lysine (K)(numbering according to Kabat) and the amino acid at position 123 issubstituted by lysine (K) or arginine (R) (numbering according to Kabat)(most particularly by arginine (R)), and wherein in the constant domainCH1 of the first antigen binding moiety under a) the amino acid atposition 147 is substituted by glutamic acid (E) (numbering according toKabat EU index) and the amino acid at position 213 is substituted byglutamic acid (E) (numbering according to Kabat EU index); andwherein the first antigen binding moiety under a) and the second antigenbinding moiety under b) are each fused at the C-terminus of the Fabheavy chain to the N-terminus of one of the subunits of the Fc domainunder c).

According to any of the above embodiments, components of the bispecificantigen binding molecule (e.g. Fab molecules, Fc domain) may be fuseddirectly or through various linkers, particularly peptide linkerscomprising one or more amino acids, typically about 2-20 amino acids,that are described herein or are known in the art. Suitable,non-immunogenic peptide linkers include, for example, (G₄S)_(n),(G₄S)_(n), or G₄(SG₄)_(n) peptide linkers, wherein n is generally aninteger from 1 to 10, typically from 2 to 4.

In a particular aspect, the invention provides a bispecific antigenbinding molecule comprising

a) a first and a third antigen binding moiety that binds to a firstantigen; wherein the first antigen is HLA-A2/WT1, particularlyHLA-A2/WT1_(RMF), and wherein the first and the second antigen bindingmoiety are each a (conventional) Fab molecule comprising a heavy chainvariable region comprising the amino acid sequence of SEQ ID NO: 7 and alight chain variable region comprising the amino acid sequence of SEQ IDNO: 8;b) a second antigen binding moiety that binds to a second antigen;wherein the second antigen is CD3 and wherein the second antigen bindingmoiety is Fab molecule wherein the variable domains VL and VH of the Fablight chain and the Fab heavy chain are replaced by each other,comprising (i) a heavy chain variable region comprising the amino acidsequence of SEQ ID NO: 121 and a light chain variable region comprisingthe amino acid sequence of SEQ ID NO: 122, or (ii) a heavy chainvariable region comprising the amino acid sequence of SEQ ID NO: 136 anda light chain variable region comprising the amino acid sequence of SEQID NO: 137 (particularly a heavy chain variable region comprising theamino acid sequence of SEQ ID NO: 136 and a light chain variable regioncomprising the amino acid sequence of SEQ ID NO: 137);c) an Fc domain composed of a first and a second subunit;whereinin the constant domain CL of the first and the third antigen bindingmoiety under a) the amino acid at position 124 is substituted by lysine(K) (numbering according to Kabat) and the amino acid at position 123 issubstituted by lysine (K) or arginine (R) (numbering according to Kabat)(most particularly by arginine (R)), and wherein in the constant domainCH1 of the first and the third antigen binding moiety under a) the aminoacid at position 147 is substituted by glutamic acid (E) (numberingaccording to Kabat EU index) and the amino acid at position 213 issubstituted by glutamic acid (E) (numbering according to Kabat EUindex);and wherein furtherthe first antigen binding moiety under a) is fused at the C-terminus ofthe Fab heavy chain to the N-terminus of the Fab heavy chain of thesecond antigen binding moiety under b), and the second antigen bindingmoiety under b) and the third antigen binding moiety under a) are eachfused at the C-terminus of the Fab heavy chain to the N-terminus of oneof the subunits of the Fc domain under c).

In a further aspect, the invention provides a bispecific antigen bindingmolecule comprising

a) a first and a third antigen binding moiety that binds to a firstantigen; wherein the first antigen is HLA-A2/WT1, particularlyHLA-A2/WT1_(RMF), and wherein the first and the second antigen bindingmoiety are each a (conventional) Fab molecule comprising a heavy chainvariable region comprising the amino acid sequence of SEQ ID NO: 15 anda light chain variable region comprising the amino acid sequence of SEQID NO: 16;b) a second antigen binding moiety that binds to a second antigen;wherein the second antigen is CD3 and wherein the second antigen bindingmoiety is Fab molecule wherein the variable domains VL and VH of the Fablight chain and the Fab heavy chain are replaced by each other,comprising (i) a heavy chain variable region comprising the amino acidsequence of SEQ ID NO: 121 and a light chain variable region comprisingthe amino acid sequence of SEQ ID NO: 122, or (ii) a heavy chainvariable region comprising the amino acid sequence of SEQ ID NO: 136 anda light chain variable region comprising the amino acid sequence of SEQID NO: 137 (particularly a heavy chain variable region comprising theamino acid sequence of SEQ ID NO: 136 and a light chain variable regioncomprising the amino acid sequence of SEQ ID NO: 137);c) an Fc domain composed of a first and a second subunit;whereinin the constant domain CL of the first and the third antigen bindingmoiety under a) the amino acid at position 124 is substituted by lysine(K) (numbering according to Kabat) and the amino acid at position 123 issubstituted by lysine (K) or arginine (R) (numbering according to Kabat)(most particularly by arginine (R)), and wherein in the constant domainCH1 of the first and the third antigen binding moiety under a) the aminoacid at position 147 is substituted by glutamic acid (E) (numberingaccording to Kabat EU index) and the amino acid at position 213 issubstituted by glutamic acid (E) (numbering according to Kabat EUindex);and wherein furtherthe first antigen binding moiety under a) is fused at the C-terminus ofthe Fab heavy chain to the N-terminus of the Fab heavy chain of thesecond antigen binding moiety under b), and the second antigen bindingmoiety under b) and the third antigen binding moiety under a) are eachfused at the C-terminus of the Fab heavy chain to the N-terminus of oneof the subunits of the Fc domain under c).

In one embodiment according to these aspects of the invention, in thefirst subunit of the Fc domain the threonine residue at position 366 isreplaced with a tryptophan residue (T366W), and in the second subunit ofthe Fc domain the tyrosine residue at position 407 is replaced with avaline residue (Y407V) and optionally the threonine residue at position366 is replaced with a serine residue (T366S) and the leucine residue atposition 368 is replaced with an alanine residue (L368A) (numberingsaccording to Kabat EU index).

In a further embodiment according to these aspects of the invention, inthe first subunit of the Fc domain additionally the serine residue atposition 354 is replaced with a cysteine residue (S354C) or the glutamicacid residue at position 356 is replaced with a cysteine residue (E356C)(particularly the serine residue at position 354 is replaced with acysteine residue), and in the second subunit of the Fc domainadditionally the tyrosine residue at position 349 is replaced by acysteine residue (Y349C) (numberings according to Kabat EU index).

In still a further embodiment according to these aspects of theinvention, in each of the first and the second subunit of the Fc domainthe leucine residue at position 234 is replaced with an alanine residue(L234A), the leucine residue at position 235 is replaced with an alanineresidue (L235A) and the proline residue at position 329 is replaced by aglycine residue (P329G) (numbering according to Kabat EU index).

In still a further embodiment according to these aspects of theinvention, the Fc domain is a human IgG₁ Fc domain.

In particular specific embodiment, the bispecific antigen bindingmolecule comprises a polypeptide comprising an amino acid sequence thatis at least 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQID NO: 123, a polypeptide comprising an amino acid sequence that is atleast 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO:125, a polypeptide comprising an amino acid sequence that is at least95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 139,and a polypeptide comprising an amino acid sequence that is at least95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 140.In a further particular specific embodiment, the bispecific antigenbinding molecule comprises a polypeptide comprising the amino acidsequence of SEQ ID NO: 123, a polypeptide comprising the amino acidsequence of SEQ ID NO: 125, a polypeptide comprising the amino acidsequence of SEQ ID NO: 139 and a polypeptide comprising the amino acidsequence of SEQ ID NO: 140.

In another specific embodiment, the bispecific antigen binding moleculecomprises a polypeptide comprising an amino acid sequence that is atleast 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO:123, a polypeptide comprising an amino acid sequence that is at least95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 124,a polypeptide comprising an amino acid sequence that is at least 95%,96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 125, and apolypeptide comprising an amino acid sequence that is at least 95%, 96%,97%, 98%, or 99% identical to the sequence of SEQ ID NO: 129. In afurther particular specific embodiment, the bispecific antigen bindingmolecule comprises a polypeptide comprising the amino acid sequence ofSEQ ID NO: 123, a polypeptide comprising the amino acid sequence of SEQID NO: 124, a polypeptide comprising the amino acid sequence of SEQ IDNO: 125 and a polypeptide comprising the amino acid sequence of SEQ IDNO: 129.

In another specific embodiment, the bispecific antigen binding moleculecomprises a polypeptide comprising an amino acid sequence that is atleast 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO:126, a polypeptide comprising an amino acid sequence that is at least95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 127,a polypeptide comprising an amino acid sequence that is at least 95%,96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 128, and apolypeptide comprising an amino acid sequence that is at least 95%, 96%,97%, 98%, or 99% identical to the sequence of SEQ ID NO: 129. In afurther specific embodiment, the bispecific antigen binding moleculecomprises a polypeptide comprising the amino acid sequence of SEQ ID NO:126, a polypeptide comprising the amino acid sequence of SEQ ID NO: 127,a polypeptide comprising the amino acid sequence of SEQ ID NO: 128 and apolypeptide comprising the amino acid sequence of SEQ ID NO: 129.

Fc Domain

In particular embodiments, the bispecific antigen binding molecule ofthe invention comprises an Fc domain composed of a first and a secondsubunit. It is understood, that the features of the Fc domain describedherein in relation to the bispecific antigen binding molecule canequally apply to an Fc domain comprised in an antibody of the invention.

The Fc domain of the bispecific antigen binding molecule consists of apair of polypeptide chains comprising heavy chain domains of animmunoglobulin molecule. For example, the Fc domain of an immunoglobulinG (IgG) molecule is a dimer, each subunit of which comprises the CH2 andCH3 IgG heavy chain constant domains. The two subunits of the Fc domainare capable of stable association with each other. In one embodiment,the bispecific antigen binding molecule of the invention comprises notmore than one Fc domain.

In one embodiment, the Fc domain of the bispecific antigen bindingmolecule is an IgG Fc domain. In a particular embodiment, the Fc domainis an IgG₁ Fc domain. In another embodiment the Fc domain is an IgG₄ Fcdomain. In a more specific embodiment, the Fc domain is an IgG₄ Fcdomain comprising an amino acid substitution at position 5228 (Kabat EUindex numbering), particularly the amino acid substitution S228P. Thisamino acid substitution reduces in vivo Fab arm exchange of IgG₄antibodies (see Stubenrauch et al., Drug Metabolism and Disposition 38,84-91 (2010)). In a further particular embodiment, the Fc domain is ahuman Fc domain. In an even more particular embodiment, the Fc domain isa human IgG₁ Fc domain. An exemplary sequence of a human IgG₁ Fc regionis given in SEQ ID NO: 109.

Fc Domain Modifications Promoting Heterodimerization

Bispecific antigen binding molecules according to the invention comprisedifferent antigen binding moieties, which may be fused to one or theother of the two subunits of the Fc domain, thus the two subunits of theFc domain are typically comprised in two non-identical polypeptidechains. Recombinant co-expression of these polypeptides and subsequentdimerization leads to several possible combinations of the twopolypeptides. To improve the yield and purity of bispecific antigenbinding molecules in recombinant production, it will thus beadvantageous to introduce in the Fc domain of the bispecific antigenbinding molecule a modification promoting the association of the desiredpolypeptides.

Accordingly, in particular embodiments, the Fc domain of the bispecificantigen binding molecule according to the invention comprises amodification promoting the association of the first and the secondsubunit of the Fc domain. The site of most extensive protein-proteininteraction between the two subunits of a human IgG Fc domain is in theCH3 domain of the Fc domain. Thus, in one embodiment said modificationis in the CH3 domain of the Fc domain.

There exist several approaches for modifications in the CH3 domain ofthe Fc domain in order to enforce heterodimerization, which are welldescribed e.g. in WO 96/27011, WO 98/050431, EP 1870459, WO 2007/110205,WO 2007/147901, WO 2009/089004, WO 2010/129304, WO 2011/90754, WO2011/143545, WO 2012058768, WO 2013157954, WO 2013096291. Typically, inall such approaches the CH3 domain of the first subunit of the Fc domainand the CH3 domain of the second subunit of the Fc domain are bothengineered in a complementary manner so that each CH3 domain (or theheavy chain comprising it) can no longer homodimerize with itself but isforced to heterodimerize with the complementarily engineered other CH3domain (so that the first and second CH3 domain heterodimerize and nohomodimers between the two first or the two second CH3 domains areformed). These different approaches for improved heavy chainheterodimerization are contemplated as different alternatives incombination with the heavy-light chain modifications (e.g. VH and VLexchange/replacement in one binding arm and the introduction ofsubstitutions of charged amino acids with opposite charges in the CH1/CLinterface) in the bispecific antigen binding molecule which reduceheavy/light chain mispairing and Bence Jones-type side products.

In a specific embodiment said modification promoting the association ofthe first and the second subunit of the Fc domain is a so-called“knob-into-hole” modification, comprising a “knob” modification in oneof the two subunits of the Fc domain and a “hole” modification in theother one of the two subunits of the Fc domain.

The knob-into-hole technology is described e.g. in U.S. Pat. Nos.5,731,168; 7,695,936; Ridgway et al., Prot Eng 9, 617-621 (1996) andCarter, J Immunol Meth 248, 7-15 (2001). Generally, the method involvesintroducing a protuberance (“knob”) at the interface of a firstpolypeptide and a corresponding cavity (“hole”) in the interface of asecond polypeptide, such that the protuberance can be positioned in thecavity so as to promote heterodimer formation and hinder homodimerformation. Protuberances are constructed by replacing small amino acidside chains from the interface of the first polypeptide with larger sidechains (e.g. tyrosine or tryptophan). Compensatory cavities of identicalor similar size to the protuberances are created in the interface of thesecond polypeptide by replacing large amino acid side chains withsmaller ones (e.g. alanine or threonine).

Accordingly, in a particular embodiment, in the CH3 domain of the firstsubunit of the Fc domain of the bispecific antigen binding molecule anamino acid residue is replaced with an amino acid residue having alarger side chain volume, thereby generating a protuberance within theCH3 domain of the first subunit which is positionable in a cavity withinthe CH3 domain of the second subunit, and in the CH3 domain of thesecond subunit of the Fc domain an amino acid residue is replaced withan amino acid residue having a smaller side chain volume, therebygenerating a cavity within the CH3 domain of the second subunit withinwhich the protuberance within the CH3 domain of the first subunit ispositionable.

Preferably said amino acid residue having a larger side chain volume isselected from the group consisting of arginine (R), phenylalanine (F),tyrosine (Y), and tryptophan (W).

Preferably said amino acid residue having a smaller side chain volume isselected from the group consisting of alanine (A), serine (S), threonine(T), and valine (V).

The protuberance and cavity can be made by altering the nucleic acidencoding the polypeptides, e.g. by site-specific mutagenesis, or bypeptide synthesis.

In a specific embodiment, in (the CH3 domain of) the first subunit ofthe Fc domain (the “knobs” subunit) the threonine residue at position366 is replaced with a tryptophan residue (T366W), and in (the CH3domain of) the second subunit of the Fc domain (the “hole” subunit) thetyrosine residue at position 407 is replaced with a valine residue(Y407V). In one embodiment, in the second subunit of the Fc domainadditionally the threonine residue at position 366 is replaced with aserine residue (T366S) and the leucine residue at position 368 isreplaced with an alanine residue (L368A) (numberings according to KabatEU index).

In yet a further embodiment, in the first subunit of the Fc domainadditionally the serine residue at position 354 is replaced with acysteine residue (S354C) or the glutamic acid residue at position 356 isreplaced with a cysteine residue (E356C) (particularly the serineresidue at position 354 is replaced with a cysteine residue), and in thesecond subunit of the Fc domain additionally the tyrosine residue atposition 349 is replaced by a cysteine residue (Y349C) (numberingsaccording to Kabat EU index). Introduction of these two cysteineresidues results in formation of a disulfide bridge between the twosubunits of the Fc domain, further stabilizing the dimer (Carter, JImmunol Methods 248, 7-15 (2001)).

In a particular embodiment, the first subunit of the Fc domain comprisesthe amino acid substitutions S354C and T366W, and the second subunit ofthe Fc domain comprises the amino acid substitutions Y349C, T366S, L368Aand Y407V (numbering according to Kabat EU index). In a particularembodiment the antigen binding moiety that binds to the second antigen(e.g. an activating T cell antigen) is fused (optionally via the firstantigen binding moiety, which binds to HLA-A2/WT1, and/or a peptidelinker) to the first subunit of the Fc domain (comprising the “knob”modification). Without wishing to be bound by theory, fusion of theantigen binding moiety that binds a second antigen, such as anactivating T cell antigen, to the knob-containing subunit of the Fcdomain will (further) minimize the generation of antigen bindingmolecules comprising two antigen binding moieties that bind to anactivating T cell antigen (steric clash of two knob-containingpolypeptides).

Other techniques of CH3-modification for enforcing theheterodimerization are contemplated as alternatives according to theinvention and are described e.g. in WO 96/27011, WO 98/050431, EP1870459, WO 2007/110205, WO 2007/147901, WO 2009/089004, WO 2010/129304,WO 2011/90754, WO 2011/143545, WO 2012/058768, WO 2013/157954, WO2013/096291.

In one embodiment, the heterodimerization approach described in EP1870459, is used alternatively. This approach is based on theintroduction of charged amino acids with opposite charges at specificamino acid positions in the CH3/CH3 domain interface between the twosubunits of the Fc domain. One preferred embodiment for the bispecificantigen binding molecule of the invention are amino acid mutationsR409D; K370E in one of the two CH3 domains (of the Fc domain) and aminoacid mutations D399K; E357K in the other one of the CH3 domains of theFc domain (numbering according to Kabat EU index).

In another embodiment, the bispecific antigen binding molecule of theinvention comprises amino acid mutation T366W in the CH3 domain of thefirst subunit of the Fc domain and amino acid mutations T366S, L368A,Y407V in the CH3 domain of the second subunit of the Fc domain, andadditionally amino acid mutations R409D; K370E in the CH3 domain of thefirst subunit of the Fc domain and amino acid mutations D399K; E357K inthe CH3 domain of the second subunit of the Fc domain (numberingsaccording to Kabat EU index).

In another embodiment, the bispecific antigen binding molecule of theinvention comprises amino acid mutations S354C, T366W in the CH3 domainof the first subunit of the Fc domain and amino acid mutations Y349C,T366S, L368A, Y407V in the CH3 domain of the second subunit of the Fcdomain, or said bispecific antigen binding molecule comprises amino acidmutations Y349C, T366W in the CH3 domain of the first subunit of the Fcdomain and amino acid mutations S354C, T366S, L368A, Y407V in the CH3domains of the second subunit of the Fc domain and additionally aminoacid mutations R409D; K370E in the CH3 domain of the first subunit ofthe Fc domain and amino acid mutations D399K; E357K in the CH3 domain ofthe second subunit of the Fc domain (all numberings according to KabatEU index).

In one embodiment, the heterodimerization approach described in WO2013/157953 is used alternatively. In one embodiment, a first CH3 domaincomprises amino acid mutation T366K and a second CH3 domain comprisesamino acid mutation L351D (numberings according to Kabat EU index). In afurther embodiment, the first CH3 domain comprises further amino acidmutation L351K. In a further embodiment, the second CH3 domain comprisesfurther an amino acid mutation selected from Y349E, Y349D and L368E(preferably L368E) (numberings according to Kabat EU index).

In one embodiment, the heterodimerization approach described in WO2012/058768 is used alternatively. In one embodiment a first CH3 domaincomprises amino acid mutations L351Y, Y407A and a second CH3 domaincomprises amino acid mutations T366A, K409F. In a further embodiment thesecond CH3 domain comprises a further amino acid mutation at positionT411, D399, 5400, F405, N390, or K392, e.g. selected from a) T411N,T411R, T411Q, T411K, T411D, T411E or T411W, b) D399R, D399W, D399Y orD399K, c) S400E, S400D, S400R, or S400K, d) F4051, F405M, F405T, F405S,F405V or F405W, e) N390R, N390K or N390D, f) K392V, K392M, K392R, K392L,K392F or K392E (numberings according to Kabat EU index). In a furtherembodiment a first CH3 domain comprises amino acid mutations L351Y,Y407A and a second CH3 domain comprises amino acid mutations T366V,K409F. In a further embodiment, a first CH3 domain comprises amino acidmutation Y407A and a second CH3 domain comprises amino acid mutationsT366A, K409F. In a further embodiment, the second CH3 domain furthercomprises amino acid mutations K392E, T411E, D399R and S400R (numberingsaccording to Kabat EU index).

In one embodiment, the heterodimerization approach described in WO2011/143545 is used alternatively, e.g. with the amino acid modificationat a position selected from the group consisting of 368 and 409(numbering according to Kabat EU index).

In one embodiment, the heterodimerization approach described in WO2011/090762, which also uses the knobs-into-holes technology describedabove, is used alternatively. In one embodiment a first CH3 domaincomprises amino acid mutation T366W and a second CH3 domain comprisesamino acid mutation Y407A. In one embodiment, a first CH3 domaincomprises amino acid mutation T366Y and a second CH3 domain comprisesamino acid mutation Y407T (numberings according to Kabat EU index).

In one embodiment, the bispecific antigen binding molecule or its Fcdomain is of IgG₂ subclass and the heterodimerization approach describedin WO 2010/129304 is used alternatively.

In an alternative embodiment, a modification promoting association ofthe first and the second subunit of the Fc domain comprises amodification mediating electrostatic steering effects, e.g. as describedin PCT publication WO 2009/089004. Generally, this method involvesreplacement of one or more amino acid residues at the interface of thetwo Fc domain subunits by charged amino acid residues so that homodimerformation becomes electrostatically unfavorable but heterodimerizationelectrostatically favorable. In one such embodiment, a first CH3 domaincomprises amino acid substitution of K392 or N392 with a negativelycharged amino acid (e.g. glutamic acid (E), or aspartic acid (D),preferably K392D or N392D) and a second CH3 domain comprises amino acidsubstitution of D399, E356, D356, or E357 with a positively chargedamino acid (e.g. lysine (K) or arginine (R), preferably D399K, E356K,D356K, or E357K, and more preferably D399K and E356K). In a furtherembodiment, the first CH3 domain further comprises amino acidsubstitution of K409 or R409 with a negatively charged amino acid (e.g.glutamic acid (E), or aspartic acid (D), preferably K409D or R409D). Ina further embodiment the first CH3 domain further or alternativelycomprises amino acid substitution of K439 and/or K370 with a negativelycharged amino acid (e.g. glutamic acid (E), or aspartic acid (D)) (allnumberings according to Kabat EU index).

In yet a further embodiment, the heterodimerization approach describedin WO 2007/147901 is used alternatively. In one embodiment, a first CH3domain comprises amino acid mutations K253E, D282K, and K322D and asecond CH3 domain comprises amino acid mutations D239K, E240K, and K292D(numberings according to Kabat EU index).

In still another embodiment, the heterodimerization approach describedin WO 2007/110205 can be used alternatively.

In one embodiment, the first subunit of the Fc domain comprises aminoacid substitutions K392D and K409D, and the second subunit of the Fcdomain comprises amino acid substitutions D356K and D399K (numberingaccording to Kabat EU index).

Fc Domain Modifications Reducing Fc Receptor Binding and/or EffectorFunction

The Fc domain confers to the bispecific antigen binding molecule (or theantibody) favorable pharmacokinetic properties, including a long serumhalf-life which contributes to good accumulation in the target tissueand a favorable tissue-blood distribution ratio. At the same time itmay, however, lead to undesirable targeting of the bispecific antigenbinding molecule (or the antibody) to cells expressing Fc receptorsrather than to the preferred antigen-bearing cells. Moreover, theco-activation of Fc receptor signaling pathways may lead to cytokinerelease which, in combination with the T cell activating properties(e.g. in embodiments of the bispecific antigen binding molecule whereinthe second antigen binding moiety binds to an activating T cell antigen)and the long half-life of the bispecific antigen binding molecule,results in excessive activation of cytokine receptors and severe sideeffects upon systemic administration. Activation of (Fcreceptor-bearing) immune cells other than T cells may even reduceefficacy of the bispecific antigen binding molecule (particularly abispecific antigen binding molecule wherein the second antigen bindingmoiety binds to an activating T cell antigen) due to the potentialdestruction of T cells e.g. by NK cells.

Accordingly, in particular embodiments, the Fc domain of the bispecificantigen binding molecule according to the invention exhibits reducedbinding affinity to an Fc receptor and/or reduced effector function, ascompared to a native IgG₁ Fc domain. In one such embodiment the Fcdomain (or the bispecific antigen binding molecule comprising said Fcdomain) exhibits less than 50%, preferably less than 20%, morepreferably less than 10% and most preferably less than 5% of the bindingaffinity to an Fc receptor, as compared to a native IgG₁ Fc domain (or abispecific antigen binding molecule comprising a native IgG₁ Fc domain),and/or less than 50%, preferably less than 20%, more preferably lessthan 10% and most preferably less than 5% of the effector function, ascompared to a native IgG₁ Fc domain domain (or a bispecific antigenbinding molecule comprising a native IgG₁ Fc domain). In one embodiment,the Fc domain domain (or the bispecific antigen binding moleculecomprising said Fc domain) does not substantially bind to an Fc receptorand/or induce effector function. In a particular embodiment the Fcreceptor is an Fcγ receptor. In one embodiment the Fc receptor is ahuman Fc receptor. In one embodiment the Fc receptor is an activating Fcreceptor. In a specific embodiment the Fc receptor is an activatinghuman Fcγ receptor, more specifically human FcγRIIIa, FcγRI or FcγRIIa,most specifically human FcγRIIIa. In one embodiment the effectorfunction is one or more selected from the group of CDC, ADCC, ADCP, andcytokine secretion. In a particular embodiment, the effector function isADCC. In one embodiment, the Fc domain domain exhibits substantiallysimilar binding affinity to neonatal Fc receptor (FcRn), as compared toa native IgG₁ Fc domain domain. Substantially similar binding to FcRn isachieved when the Fc domain (or the bispecific antigen binding moleculecomprising said Fc domain) exhibits greater than about 70%, particularlygreater than about 80%, more particularly greater than about 90% of thebinding affinity of a native IgG₁ Fc domain (or the bispecific antigenbinding molecule comprising a native IgG₁ Fc domain) to FcRn.

In certain embodiments the Fc domain is engineered to have reducedbinding affinity to an Fc receptor and/or reduced effector function, ascompared to a non-engineered Fc domain. In particular embodiments, theFc domain of the bispecific antigen binding molecule comprises one ormore amino acid mutation that reduces the binding affinity of the Fcdomain to an Fc receptor and/or effector function. Typically, the sameone or more amino acid mutation is present in each of the two subunitsof the Fc domain. In one embodiment, the amino acid mutation reduces thebinding affinity of the Fc domain to an Fc receptor. In one embodiment,the amino acid mutation reduces the binding affinity of the Fc domain toan Fc receptor by at least 2-fold, at least 5-fold, or at least 10-fold.In embodiments where there is more than one amino acid mutation thatreduces the binding affinity of the Fc domain to the Fc receptor, thecombination of these amino acid mutations may reduce the bindingaffinity of the Fc domain to an Fc receptor by at least 10-fold, atleast 20-fold, or even at least 50-fold. In one embodiment thebispecific antigen binding molecule comprising an engineered Fc domainexhibits less than 20%, particularly less than 10%, more particularlyless than 5% of the binding affinity to an Fc receptor as compared to abispecific antigen binding molecule comprising a non-engineered Fcdomain. In a particular embodiment, the Fc receptor is an Fcγ receptor.In some embodiments, the Fc receptor is a human Fc receptor.

In some embodiments, the Fc receptor is an activating Fc receptor. In aspecific embodiment, the Fc receptor is an activating human Fcγreceptor, more specifically human FcγRIIIa, FcγRI or FcγRIIa, mostspecifically human FcγRIIIa. Preferably, binding to each of thesereceptors is reduced. In some embodiments, binding affinity to acomplement component, specifically binding affinity to C1q, is alsoreduced. In one embodiment, binding affinity to neonatal Fc receptor(FcRn) is not reduced. Substantially similar binding to FcRn, i.e.preservation of the binding affinity of the Fc domain to said receptor,is achieved when the Fc domain (or the bispecific antigen bindingmolecule comprising said Fc domain) exhibits greater than about 70% ofthe binding affinity of a non-engineered form of the Fc domain (or thebispecific antigen binding molecule comprising said non-engineered formof the Fc domain) to FcRn. The Fc domain, or bispecific antigen bindingmolecules of the invention comprising said Fc domain, may exhibitgreater than about 80% and even greater than about 90% of such affinity.In certain embodiments, the Fc domain of the bispecific antigen bindingmolecule is engineered to have reduced effector function, as compared toa non-engineered Fc domain. The reduced effector function can include,but is not limited to, one or more of the following: reduced complementdependent cytotoxicity (CDC), reduced antibody-dependent cell-mediatedcytotoxicity (ADCC), reduced antibody-dependent cellular phagocytosis(ADCP), reduced cytokine secretion, reduced immune complex-mediatedantigen uptake by antigen-presenting cells, reduced binding to NK cells,reduced binding to macrophages, reduced binding to monocytes, reducedbinding to polymorphonuclear cells, reduced direct signaling inducingapoptosis, reduced crosslinking of target-bound antibodies, reduceddendritic cell maturation, or reduced T cell priming. In one embodiment,the reduced effector function is one or more selected from the group ofreduced CDC, reduced ADCC, reduced ADCP, and reduced cytokine secretion.In a particular embodiment, the reduced effector function is reducedADCC. In one embodiment the reduced ADCC is less than 20% of the ADCCinduced by a non-engineered Fc domain (or a bispecific antigen bindingmolecule comprising a non-engineered Fc domain).

In one embodiment, the amino acid mutation that reduces the bindingaffinity of the Fc domain to an Fc receptor and/or effector function isan amino acid substitution. In one embodiment, the Fc domain comprisesan amino acid substitution at a position selected from the group ofE233, L234, L235, N297, P331 and P329 (numberings according to Kabat EUindex). In a more specific embodiment, the Fc domain comprises an aminoacid substitution at a position selected from the group of L234, L235and P329 (numberings according to Kabat EU index). In some embodiments,the Fc domain comprises the amino acid substitutions L234A and L235A(numberings according to Kabat EU index). In one such embodiment, the Fcdomain is an IgG₁ Fc domain, particularly a human IgG₁ Fc domain. In oneembodiment, the Fc domain comprises an amino acid substitution atposition P329. In a more specific embodiment, the amino acidsubstitution is P329A or P329G, particularly P329G (numberings accordingto Kabat EU index). In one embodiment, the Fc domain comprises an aminoacid substitution at position P329 and a further amino acid substitutionat a position selected from E233, L234, L235, N297 and P331 (numberingsaccording to Kabat EU index). In a more specific embodiment, the furtheramino acid substitution is E233P, L234A, L235A, L235E, N297A, N297D orP331S. In particular embodiments, the Fc domain comprises amino acidsubstitutions at positions P329, L234 and L235 (numberings according toKabat EU index). In more particular embodiments, the Fc domain comprisesthe amino acid mutations L234A, L235A and P329G (“P329G LALA”, “PGLALA”or “LALAPG”). Specifically, in particular embodiments, each subunit ofthe Fc domain comprises the amino acid substitutions L234A, L235A andP329G (Kabat EU index numbering), i.e. in each of the first and thesecond subunit of the Fc domain the leucine residue at position 234 isreplaced with an alanine residue (L234A), the leucine residue atposition 235 is replaced with an alanine residue (L235A) and the prolineresidue at position 329 is replaced by a glycine residue (P329G)(numbering according to Kabat EU index).

In one such embodiment, the Fc domain is an IgG₁ Fc domain, particularlya human IgG₁ Fc domain. The “P329G LALA” combination of amino acidsubstitutions almost completely abolishes Fcγ receptor (as well ascomplement) binding of a human IgG₁ Fc domain, as described in PCTpublication no. WO 2012/130831, which is incorporated herein byreference in its entirety. WO 2012/130831 also describes methods ofpreparing such mutant Fc domains and methods for determining itsproperties such as Fc receptor binding or effector functions.

IgG₄ antibodies exhibit reduced binding affinity to Fc receptors andreduced effector functions as compared to IgG₁ antibodies. Hence, insome embodiments, the Fc domain of the bispecific antigen bindingmolecules of the invention is an IgG₄ Fc domain, particularly a humanIgG₄ Fc domain. In one embodiment, the IgG₄ Fc domain comprises aminoacid substitutions at position 5228, specifically the amino acidsubstitution S228P (numberings according to Kabat EU index). To furtherreduce its binding affinity to an Fc receptor and/or its effectorfunction, in one embodiment, the IgG₄ Fc domain comprises an amino acidsubstitution at position L235, specifically the amino acid substitutionL235E (numberings according to Kabat EU index). In another embodiment,the IgG₄ Fc domain comprises an amino acid substitution at positionP329, specifically the amino acid substitution P329G (numberingsaccording to Kabat EU index). In a particular embodiment, the IgG₄ Fcdomain comprises amino acid substitutions at positions S228, L235 andP329, specifically amino acid substitutions S228P, L235E and P329G(numberings according to Kabat EU index). Such IgG₄ Fc domain mutantsand their Fcγ receptor binding properties are described in PCTpublication no. WO 2012/130831, incorporated herein by reference in itsentirety.

In a particular embodiment, the Fc domain exhibiting reduced bindingaffinity to an Fc receptor and/or reduced effector function, as comparedto a native IgG₁ Fc domain, is a human IgG₁ Fc domain comprising theamino acid substitutions L234A, L235A and optionally P329G, or a humanIgG₄ Fc domain comprising the amino acid substitutions S228P, L235E andoptionally P329G (numberings according to Kabat EU index).

In certain embodiments, N-glycosylation of the Fc domain has beeneliminated. In one such embodiment, the Fc domain comprises an aminoacid mutation at position N297, particularly an amino acid substitutionreplacing asparagine by alanine (N297A) or aspartic acid (N297D)(numberings according to Kabat EU index).

In addition to the Fc domains described hereinabove and in PCTpublication no. WO 2012/130831, Fc domains with reduced Fc receptorbinding and/or effector function also include those with substitution ofone or more of Fc domain residues 238, 265, 269, 270, 297, 327 and 329(U.S. Pat. No. 6,737,056) (numberings according to Kabat EU index). SuchFc mutants include Fc mutants with substitutions at two or more of aminoacid positions 265, 269, 270, 297 and 327, including the so-called“DANA” Fc mutant with substitution of residues 265 and 297 to alanine(U.S. Pat. No. 7,332,581).

Mutant Fc domains can be prepared by amino acid deletion, substitution,insertion or modification using genetic or chemical methods well knownin the art. Genetic methods may include site-specific mutagenesis of theencoding DNA sequence, PCR, gene synthesis, and the like. The correctnucleotide changes can be verified for example by sequencing.

Binding to Fc receptors can be easily determined e.g. by ELISA, or bySurface Plasmon Resonance (SPR) using standard instrumentation such as aBIAcore instrument (GE Healthcare), and Fc receptors such as may beobtained by recombinant expression. Alternatively, binding affinity ofFc domains or bispecific antigen binding molecules comprising an Fcdomain for Fc receptors may be evaluated using cell lines known toexpress particular Fc receptors, such as human NK cells expressingFcγIIIa receptor.

Effector function of an Fc domain, or a bispecific antigen bindingmolecule comprising an Fc domain, can be measured by methods known inthe art. Examples of in vitro assays to assess ADCC activity of amolecule of interest are described in U.S. Pat. No. 5,500,362; Hellstromet al. Proc Natl Acad Sci USA 83, 7059-7063 (1986) and Hellstrom et al.,Proc Natl Acad Sci USA 82, 1499-1502 (1985); U.S. Pat. No. 5,821,337;Bruggemann et al., J Exp Med 166, 1351-1361 (1987). Alternatively,non-radioactive assays methods may be employed (see, for example, ACTI™non-radioactive cytotoxicity assay for flow cytometry (CellTechnology,Inc. Mountain View, Calif.); and CytoTox 96® non-radioactivecytotoxicity assay (Promega, Madison, Wis.)). Useful effector cells forsuch assays include peripheral blood mononuclear cells (PBMC) andNatural Killer (NK) cells. Alternatively, or additionally, ADCC activityof the molecule of interest may be assessed in vivo, e.g. in a animalmodel such as that disclosed in Clynes et al., Proc Natl Acad Sci USA95, 652-656 (1998).

In some embodiments, binding of the Fc domain to a complement component,specifically to C1q, is reduced. Accordingly, in some embodimentswherein the Fc domain is engineered to have reduced effector function,said reduced effector function includes reduced CDC. C1q binding assaysmay be carried out to determine whether the Fc domain, or the bispecificantigen binding molecule comprising the Fc domain, is able to bind C1qand hence has CDC activity. See e.g., C1q and C3c binding ELISA in WO2006/029879 and WO 2005/100402. To assess complement activation, a CDCassay may be performed (see, for example, Gazzano-Santoro et al., JImmunol Methods 202, 163 (1996); Cragg et al., Blood 101, 1045-1052(2003); and Cragg and Glennie, Blood 103, 2738-2743 (2004)).

FcRn binding and in vivo clearance/half life determinations can also beperformed using methods known in the art (see, e.g., Petkova, S. B. etal., Int'l. Immunol. 18(12):1759-1769 (2006); WO 2013/120929).

Polynucleotides

The invention further provides isolated polynucleotides encoding anantibody or bispecific antigen binding molecule as described herein or afragment thereof. In some embodiments, said fragment is an antigenbinding fragment.

The polynucleotides encoding antibodies or bispecific antigen bindingmolecules of the invention may be expressed as a single polynucleotidethat encodes the entire antibody or bispecific antigen binding moleculeor as multiple (e.g., two or more) polynucleotides that areco-expressed. Polypeptides encoded by polynucleotides that areco-expressed may associate through, e.g., disulfide bonds or other meansto form a functional antibody or bispecific antigen binding molecule.For example, the light chain portion of an antibody or bispecificantigen binding molecule may be encoded by a separate polynucleotidefrom the portion of the antibody or bispecific antigen binding moleculecomprising the heavy chain of the antibody or bispecific antigen bindingmolecule. When co-expressed, the heavy chain polypeptides will associatewith the light chain polypeptides to form the antibody or bispecificantigen binding molecule. In another example, the portion of theantibody or bispecific antigen binding molecule comprising one of thetwo Fc domain subunits and optionally (part of) one or more Fabmolecules could be encoded by a separate polynucleotide from the portionof the antibody or bispecific antigen binding molecule comprising theother of the two Fc domain subunits and optionally (part of) a Fabmolecule. When co-expressed, the Fc domain subunits will associate toform the Fc domain.

In some embodiments, the isolated polynucleotide encodes the entireantibody or bispecific antigen binding molecule according to theinvention as described herein. In other embodiments, the isolatedpolynucleotide encodes a polypeptide comprised in the antibody orbispecific antigen binding molecule according to the invention asdescribed herein.

In certain embodiments the polynucleotide or nucleic acid is DNA. Inother embodiments, a polynucleotide of the present invention is RNA, forexample, in the form of messenger RNA (mRNA). RNA of the presentinvention may be single stranded or double stranded.

Recombinant Methods

Antibodies or bispecific antigen binding molecules of the invention maybe obtained, for example, by solid-state peptide synthesis (e.g.Merrifield solid phase synthesis) or recombinant production. Forrecombinant production one or more polynucleotide encoding the antibodyor bispecific antigen binding molecule (fragment), e.g., as describedabove, is isolated and inserted into one or more vectors for furthercloning and/or expression in a host cell. Such polynucleotide may bereadily isolated and sequenced using conventional procedures. In oneembodiment a vector, preferably an expression vector, comprising one ormore of the polynucleotides of the invention is provided. Methods whichare well known to those skilled in the art can be used to constructexpression vectors containing the coding sequence of an antibody orbispecific antigen binding molecule (fragment) along with appropriatetranscriptional/translational control signals. These methods include invitro recombinant DNA techniques, synthetic techniques and in vivorecombination/genetic recombination. See, for example, the techniquesdescribed in Maniatis et al., MOLECULAR CLONING: A LABORATORY MANUAL,Cold Spring Harbor Laboratory, N.Y. (1989); and Ausubel et al., CURRENTPROTOCOLS IN MOLECULAR BIOLOGY, Greene Publishing Associates and WileyInterscience, N.Y (1989). The expression vector can be part of aplasmid, virus, or may be a nucleic acid fragment. The expression vectorincludes an expression cassette into which the polynucleotide encodingthe antibody or bispecific antigen binding molecule (fragment) (i.e. thecoding region) is cloned in operable association with a promoter and/orother transcription or translation control elements. As used herein, a“coding region” is a portion of nucleic acid which consists of codonstranslated into amino acids. Although a “stop codon” (TAG, TGA, or TAA)is not translated into an amino acid, it may be considered to be part ofa coding region, if present, but any flanking sequences, for examplepromoters, ribosome binding sites, transcriptional terminators, introns,5′ and 3′ untranslated regions, and the like, are not part of a codingregion. Two or more coding regions can be present in a singlepolynucleotide construct, e.g. on a single vector, or in separatepolynucleotide constructs, e.g. on separate (different) vectors.Furthermore, any vector may contain a single coding region, or maycomprise two or more coding regions, e.g. a vector of the presentinvention may encode one or more polypeptides, which are post- orco-translationally separated into the final proteins via proteolyticcleavage. In addition, a vector, polynucleotide, or nucleic acid of theinvention may encode heterologous coding regions, either fused orunfused to a polynucleotide encoding the antibody or bispecific antigenbinding molecule (fragment) of the invention, or variant or derivativethereof. Heterologous coding regions include without limitationspecialized elements or motifs, such as a secretory signal peptide or aheterologous functional domain. An operable association is when a codingregion for a gene product, e.g. a polypeptide, is associated with one ormore regulatory sequences in such a way as to place expression of thegene product under the influence or control of the regulatorysequence(s). Two DNA fragments (such as a polypeptide coding region anda promoter associated therewith) are “operably associated” if inductionof promoter function results in the transcription of mRNA encoding thedesired gene product and if the nature of the linkage between the twoDNA fragments does not interfere with the ability of the expressionregulatory sequences to direct the expression of the gene product orinterfere with the ability of the DNA template to be transcribed. Thus,a promoter region would be operably associated with a nucleic acidencoding a polypeptide if the promoter was capable of effectingtranscription of that nucleic acid. The promoter may be a cell-specificpromoter that directs substantial transcription of the DNA only inpredetermined cells. Other transcription control elements, besides apromoter, for example enhancers, operators, repressors, andtranscription termination signals, can be operably associated with thepolynucleotide to direct cell-specific transcription. Suitable promotersand other transcription control regions are disclosed herein. A varietyof transcription control regions are known to those skilled in the art.These include, without limitation, transcription control regions, whichfunction in vertebrate cells, such as, but not limited to, promoter andenhancer segments from cytomegaloviruses (e.g. the immediate earlypromoter, in conjunction with intron-A), simian virus 40 (e.g. the earlypromoter), and retroviruses (such as, e.g. Rous sarcoma virus). Othertranscription control regions include those derived from vertebrategenes such as actin, heat shock protein, bovine growth hormone andrabbit β-globin, as well as other sequences capable of controlling geneexpression in eukaryotic cells. Additional suitable transcriptioncontrol regions include tissue-specific promoters and enhancers as wellas inducible promoters (e.g. promoters inducible tetracyclins).Similarly, a variety of translation control elements are known to thoseof ordinary skill in the art. These include, but are not limited toribosome binding sites, translation initiation and termination codons,and elements derived from viral systems (particularly an internalribosome entry site, or IRES, also referred to as a CITE sequence). Theexpression cassette may also include other features such as an origin ofreplication, and/or chromosome integration elements such as retrovirallong terminal repeats (LTRs), or adeno-associated viral (AAV) invertedterminal repeats (ITRs).

Polynucleotide and nucleic acid coding regions of the present inventionmay be associated with additional coding regions which encode secretoryor signal peptides, which direct the secretion of a polypeptide encodedby a polynucleotide of the present invention. For example, if secretionof the antibody or bispecific antigen binding molecule is desired, DNAencoding a signal sequence may be placed upstream of the nucleic acidencoding an antibody or bispecific antigen binding molecule of theinvention or a fragment thereof. According to the signal hypothesis,proteins secreted by mammalian cells have a signal peptide or secretoryleader sequence which is cleaved from the mature protein once export ofthe growing protein chain across the rough endoplasmic reticulum hasbeen initiated. Those of ordinary skill in the art are aware thatpolypeptides secreted by vertebrate cells generally have a signalpeptide fused to the N-terminus of the polypeptide, which is cleavedfrom the translated polypeptide to produce a secreted or “mature” formof the polypeptide. In certain embodiments, the native signal peptide,e.g. an immunoglobulin heavy chain or light chain signal peptide isused, or a functional derivative of that sequence that retains theability to direct the secretion of the polypeptide that is operablyassociated with it. Alternatively, a heterologous mammalian signalpeptide, or a functional derivative thereof, may be used. For example,the wild-type leader sequence may be substituted with the leadersequence of human tissue plasminogen activator (TPA) or mouseβ-glucuronidase.

DNA encoding a short protein sequence that could be used to facilitatelater purification (e.g. a histidine tag) or assist in labeling theantibody or bispecific antigen binding molecule may be included withinor at the ends of the antibody or bispecific antigen binding molecule(fragment) encoding polynucleotide.

In a further embodiment, a host cell comprising one or morepolynucleotides of the invention is provided. In certain embodiments ahost cell comprising one or more vectors of the invention is provided.The polynucleotides and vectors may incorporate any of the features,singly or in combination, described herein in relation topolynucleotides and vectors, respectively. In one such embodiment a hostcell comprises (e.g. has been transformed or transfected with) one ormore vector comprising one or more polynucleotide that encodes (part of)an antibody or bispecific antigen binding molecule of the invention. Asused herein, the term “host cell” refers to any kind of cellular systemwhich can be engineered to generate the antibody or bispecific antigenbinding molecule of the invention or fragments thereof. Host cellssuitable for replicating and for supporting expression of antibodies orbispecific antigen binding molecules are well known in the art. Suchcells may be transfected or transduced as appropriate with theparticular expression vector and large quantities of vector containingcells can be grown for seeding large scale fermenters to obtainsufficient quantities of the antibody or bispecific antigen bindingmolecule for clinical applications. Suitable host cells includeprokaryotic microorganisms, such as E. coli, or various eukaryoticcells, such as Chinese hamster ovary cells (CHO), insect cells, or thelike. For example, polypeptides may be produced in bacteria inparticular when glycosylation is not needed. After expression, thepolypeptide may be isolated from the bacterial cell paste in a solublefraction and can be further purified. In addition to prokaryotes,eukaryotic microbes such as filamentous fungi or yeast are suitablecloning or expression hosts for polypeptide-encoding vectors, includingfungi and yeast strains whose glycosylation pathways have been“humanized”, resulting in the production of a polypeptide with apartially or fully human glycosylation pattern. See Gerngross, NatBiotech 22, 1409-1414 (2004), and Li et al., Nat Biotech 24, 210-215(2006). Suitable host cells for the expression of (glycosylated)polypeptides are also derived from multicellular organisms(invertebrates and vertebrates). Examples of invertebrate cells includeplant and insect cells. Numerous baculoviral strains have beenidentified which may be used in conjunction with insect cells,particularly for transfection of Spodoptera frugiperda cells. Plant cellcultures can also be utilized as hosts. See e.g. U.S. Pat. Nos.5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (describingPLANTIBODIES™ technology for producing antibodies in transgenic plants).Vertebrate cells may also be used as hosts. For example, mammalian celllines that are adapted to grow in suspension may be useful. Otherexamples of useful mammalian host cell lines are monkey kidney CV1 linetransformed by SV40 (COS-7); human embryonic kidney line (293 or 293Tcells as described, e.g., in Graham et al., J Gen Virol 36, 59 (1977)),baby hamster kidney cells (BHK), mouse sertoli cells (TM4 cells asdescribed, e.g., in Mather, Biol Reprod 23, 243-251 (1980)), monkeykidney cells (CV1), African green monkey kidney cells (VERO-76), humancervical carcinoma cells (HELA), canine kidney cells (MDCK), buffalo ratliver cells (BRL 3A), human lung cells (W138), human liver cells (HepG2), mouse mammary tumor cells (MMT 060562), TRI cells (as described,e.g., in Mather et al., Annals N.Y. Acad Sci 383, 44-68 (1982)), MRC 5cells, and FS4 cells. Other useful mammalian host cell lines includeChinese hamster ovary (CHO) cells, including dhfr CHO cells (Urlaub etal., Proc Natl Acad Sci USA 77, 4216 (1980)); and myeloma cell linessuch as YO, NS0, P3X63 and Sp2/0. For a review of certain mammalian hostcell lines suitable for protein production, see, e.g., Yazaki and Wu,Methods in Molecular Biology, Vol. 248 (B. K. C. Lo, ed., Humana Press,Totowa, N.J.), pp. 255-268 (2003). Host cells include cultured cells,e.g., mammalian cultured cells, yeast cells, insect cells, bacterialcells and plant cells, to name only a few, but also cells comprisedwithin a transgenic animal, transgenic plant or cultured plant or animaltissue. In one embodiment, the host cell is a eukaryotic cell,preferably a mammalian cell, such as a Chinese Hamster Ovary (CHO) cell,a human embryonic kidney (HEK) cell or a lymphoid cell (e.g., Y0, NS0,Sp20 cell).

Standard technologies are known in the art to express foreign genes inthese systems. Cells expressing a polypeptide comprising either theheavy or the light chain of an antigen binding domain such as anantibody, may be engineered so as to also express the other of theantibody chains such that the expressed product is an antibody that hasboth a heavy and a light chain.

In one embodiment, a method of producing an antibody or bispecificantigen binding molecule according to the invention is provided, whereinthe method comprises culturing a host cell comprising a polynucleotideencoding the antibody or bispecific antigen binding molecule, asprovided herein, under conditions suitable for expression of theantibody or bispecific antigen binding molecule, and optionallyrecovering the antibody or bispecific antigen binding molecule from thehost cell (or host cell culture medium).

The components of the bispecific antigen binding molecule (or theantibody) of the invention may be genetically fused to each other. Thebispecific antigen binding molecule can be designed such that itscomponents are fused directly to each other or indirectly through alinker sequence. The composition and length of the linker may bedetermined in accordance with methods well known in the art and may betested for efficacy. Examples of linker sequences between differentcomponents of bispecific antigen binding molecules are provided herein.Additional sequences may also be included to incorporate a cleavage siteto separate the individual components of the fusion if desired, forexample an endopeptidase recognition sequence.

The antibody or bispecific antigen binding molecule of the inventiongenerally comprise at least an antibody variable region capable ofbinding an antigenic determinant. Variable regions can form part of andbe derived from naturally or non-naturally occurring antibodies andfragments thereof. Methods to produce polyclonal antibodies andmonoclonal antibodies are well known in the art (see e.g. Harlow andLane, “Antibodies, a laboratory manual”, Cold Spring Harbor Laboratory,1988). Non-naturally occurring antibodies can be constructed using solidphase-peptide synthesis, can be produced recombinantly (e.g. asdescribed in U.S. Pat. No. 4,186,567) or can be obtained, for example,by screening combinatorial libraries comprising variable heavy chainsand variable light chains (see e.g. U.S. Pat. No. 5,969,108 toMcCafferty).

Any animal species of antibody, antibody fragment, antigen bindingdomain or variable region may be used in the antibody or bispecificantigen binding molecule of the invention. Non-limiting antibodies,antibody fragments, antigen binding domains or variable regions usefulin the present invention can be of murine, primate, or human origin. Ifthe antibody or bispecific antigen binding molecule is intended forhuman use, a chimeric form of antibody may be used wherein the constantregions of the antibody are from a human. A humanized or fully humanform of the antibody can also be prepared in accordance with methodswell known in the art (see e. g. U.S. Pat. No. 5,565,332 to Winter).Humanization may be achieved by various methods including, but notlimited to (a) grafting the non-human (e.g., donor antibody) CDRs ontohuman (e.g. recipient antibody) framework and constant regions with orwithout retention of critical framework residues (e.g. those that areimportant for retaining good antigen binding affinity or antibodyfunctions), (b) grafting only the non-human specificity-determiningregions (SDRs or a-CDRs; the residues critical for the antibody-antigeninteraction) onto human framework and constant regions, or (c)transplanting the entire non-human variable domains, but “cloaking” themwith a human-like section by replacement of surface residues. Humanizedantibodies and methods of making them are reviewed, e.g., in Almagro andFransson, Front. Biosci. 13:1619-1633 (2008), and are further described,e.g., in Riechmann et al., Nature 332:323-329 (1988); Queen et al.,Proc. Nat'l Acad. Sci. USA 86:10029-10033 (1989); U.S. Pat. Nos.5,821,337, 7,527,791, 6,982,321, and 7,087,409; Kashmiri et al., Methods36:25-34 (2005) (describing specificity determining region (SDR)grafting); Padlan, Mol. Immunol. 28:489-498 (1991) (describing“resurfacing”); Dall'Acqua et al., Methods 36:43-60 (2005) (describing“FR shuffling”); and Osbourn et al., Methods 36:61-68 (2005) and Klimkaet al., Br. J. Cancer, 83:252-260 (2000) (describing the “guidedselection” approach to FR shuffling). Human framework regions that maybe used for humanization include but are not limited to: frameworkregions selected using the “best-fit” method (see, e.g., Sims et al. J.Immunol. 151:2296 (1993)); framework regions derived from the consensussequence of human antibodies of a particular subgroup of light or heavychain variable regions (see, e.g., Carter et al. Proc. Natl. Acad. Sci.USA, 89:4285 (1992); and Presta et al. J. Immunol., 151:2623 (1993));human mature (somatically mutated) framework regions or human germlineframework regions (see, e.g., Almagro and Fransson, Front. Biosci.13:1619-1633 (2008)); and framework regions derived from screening FRlibraries (see, e.g., Baca et al., J. Biol. Chem. 272:10678-10684 (1997)and Rosok et al., J. Biol. Chem. 271:22611-22618 (1996)).

Human antibodies can be produced using various techniques known in theart. Human antibodies are described generally in van Dijk and van deWinkel, Curr Opin Pharmacol 5, 368-74 (2001) and Lonberg, Curr OpinImmunol 20, 450-459 (2008). Human antibodies may be prepared byadministering an immunogen to a transgenic animal that has been modifiedto produce intact human antibodies or intact antibodies with humanvariable regions in response to antigenic challenge. Such animalstypically contain all or a portion of the human immunoglobulin loci,which replace the endogenous immunoglobulin loci, or which are presentextrachromosomally or integrated randomly into the animal's chromosomes.In such transgenic mice, the endogenous immunoglobulin loci havegenerally been inactivated. For review of methods for obtaining humanantibodies from transgenic animals, see Lonberg, Nat. Biotech.23:1117-1125 (2005). See also, e.g., U.S. Pat. Nos. 6,075,181 and6,150,584 describing XENOMOUSE™ technology; U.S. Pat. No. 5,770,429describing HUMAB® technology; U.S. Pat. No. 7,041,870 describing K-MMOUSE® technology, and U.S. Patent Application Publication No. US2007/0061900, describing VELOCIMOUSE® technology). Human variableregions from intact antibodies generated by such animals may be furthermodified, e.g., by combining with a different human constant region.

Human antibodies can also be made by hybridoma-based methods. Humanmyeloma and mouse-human heteromyeloma cell lines for the production ofhuman monoclonal antibodies have been described. (See, e.g., Kozbor J.Immunol., 133: 3001 (1984); Brodeur et al., Monoclonal AntibodyProduction Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc.,New York, 1987); and Boerner et al., J. Immunol., 147: 86 (1991).) Humanantibodies generated via human B-cell hybridoma technology are alsodescribed in Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562(2006). Additional methods include those described, for example, in U.S.Pat. No. 7,189,826 (describing production of monoclonal human IgMantibodies from hybridoma cell lines) and Ni, Xiandai Mianyixue,26(4):265-268 (2006) (describing human-human hybridomas). Humanhybridoma technology (Trioma technology) is also described in Vollmersand Brandlein, Histology and Histopathology, 20(3):927-937 (2005) andVollmers and Brandlein, Methods and Findings in Experimental andClinical Pharmacology, 27(3):185-91 (2005).

Human antibodies may also be generated by isolation from human antibodylibraries, as described herein.

Antibodies useful in the invention may be isolated by screeningcombinatorial libraries for antibodies with the desired activity oractivities. Methods for screening combinatorial libraries are reviewed,e.g., in Lerner et al. in Nature Reviews 16:498-508 (2016). For example,a variety of methods are known in the art for generating phage displaylibraries and screening such libraries for antibodies possessing thedesired binding characteristics. Such methods are reviewed, e.g., inFrenzel et al. in mAbs 8:1177-1194 (2016); Bazan et al. in HumanVaccines and Immunotherapeutics 8:1817-1828 (2012) and Zhao et al. inCritical Reviews in Biotechnology 36:276-289 (2016) as well as inHoogenboom et al. in Methods in Molecular Biology 178:1-37 (O'Brien etal., ed., Human Press, Totowa, N.J., 2001) and in Marks and Bradbury inMethods in Molecular Biology 248:161-175 (Lo, ed., Human Press, Totowa,N.J., 2003).

In certain phage display methods, repertoires of VH and VL genes areseparately cloned by polymerase chain reaction (PCR) and recombinedrandomly in phage libraries, which can then be screened forantigen-binding phage as described in Winter et al. in Annual Review ofImmunology 12: 433-455 (1994). Phage typically display antibodyfragments, either as single-chain Fv (scFv) fragments or as Fabfragments. Libraries from immunized sources provide high-affinityantibodies to the immunogen without the requirement of constructinghybridomas. Alternatively, the naive repertoire can be cloned (e.g.,from human) to provide a single source of antibodies to a wide range ofnon-self and also self antigens without any immunization as described byGriffiths et al. in EMBO Journal 12: 725-734 (1993). Finally, naivelibraries can also be made synthetically by cloning unrearranged V-genesegments from stem cells, and using PCR primers containing randomsequence to encode the highly variable CDR3 regions and to accomplishrearrangement in vitro, as described by Hoogenboom and Winter in Journalof Molecular Biology 227: 381-388 (1992). Patent publications describinghuman antibody phage libraries include, for example: U.S. Pat. Nos.5,750,373; 7,985,840; 7,785,903 and 8,679,490 as well as US PatentPublication Nos. 2005/0079574, 2007/0117126, 2007/0237764 and2007/0292936. Further examples of methods known in the art for screeningcombinatorial libraries for antibodies with a desired activity oractivities include ribosome and mRNA display, as well as methods forantibody display and selection on bacteria, mammalian cells, insectcells or yeast cells. Methods for yeast surface display are reviewed,e.g., in Scholler et al. in Methods in Molecular Biology 503:135-56(2012) and in Cherf et al. in Methods in Molecular biology 1319:155-175(2015) as well as in the Zhao et al. in Methods in Molecular Biology889:73-84 (2012). Methods for ribosome display are described, e.g., inHe et al. in Nucleic Acids Research 25:5132-5134 (1997) and in Hanes etal. in PNAS 94:4937-4942 (1997).

Antibodies or bispecific antigen binding molecules prepared as describedherein may be purified by art-known techniques such as high performanceliquid chromatography, ion exchange chromatography, gel electrophoresis,affinity chromatography, size exclusion chromatography, and the like.The actual conditions used to purify a particular protein will depend,in part, on factors such as net charge, hydrophobicity, hydrophilicityetc., and will be apparent to those having skill in the art. Foraffinity chromatography purification, an antibody, ligand, receptor orantigen can be used to which the antibody or bispecific antigen bindingmolecule binds. For example, for affinity chromatography purification ofantibodies or bispecific antigen binding molecules of the invention, amatrix with protein A or protein G may be used. Sequential Protein A orG affinity chromatography and size exclusion chromatography can be usedto isolate an antibody or bispecific antigen binding moleculeessentially as described in the Examples. The purity of the antibody orbispecific antigen binding molecule can be determined by any of avariety of well known analytical methods including gel electrophoresis,high pressure liquid chromatography, and the like.

Compositions, Formulations, and Routes of Administration

In a further aspect, the invention provides pharmaceutical compositionscomprising any of the antibodies or bispecific antigen binding moleculesprovided herein, e.g., for use in any of the below therapeutic methods.In one embodiment, a pharmaceutical composition comprises any of theantibodies or bispecific antigen binding molecules provided herein and apharmaceutically acceptable carrier. In another embodiment, apharmaceutical composition comprises any of the antibodies or bispecificantigen binding molecules provided herein and at least one additionaltherapeutic agent, e.g., as described below.

Further provided is a method of producing an antibody or bispecificantigen binding molecule of the invention in a form suitable foradministration in vivo, the method comprising (a) obtaining an antibodyor bispecific antigen binding molecule according to the invention, and(b) formulating the antibody or bispecific antigen binding molecule withat least one pharmaceutically acceptable carrier, whereby a preparationof antibody or bispecific antigen binding molecule is formulated foradministration in vivo.

Pharmaceutical compositions of the present invention comprise atherapeutically effective amount of antibody or bispecific antigenbinding molecule dissolved or dispersed in a pharmaceutically acceptablecarrier. The phrases “pharmaceutical or pharmacologically acceptable”refers to molecular entities and compositions that are generallynon-toxic to recipients at the dosages and concentrations employed, i.e.do not produce an adverse, allergic or other untoward reaction whenadministered to an animal, such as, for example, a human, asappropriate. The preparation of a pharmaceutical composition thatcontains an antibody or bispecific antigen binding molecule andoptionally an additional active ingredient will be known to those ofskill in the art in light of the present disclosure, as exemplified byRemington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company,1990, incorporated herein by reference. Moreover, for animal (e.g.,human) administration, it will be understood that preparations shouldmeet sterility, pyrogenicity, general safety and purity standards asrequired by FDA Office of Biological Standards or correspondingauthorities in other countries. Preferred compositions are lyophilizedformulations or aqueous solutions. As used herein, “pharmaceuticallyacceptable carrier” includes any and all solvents, buffers, dispersionmedia, coatings, surfactants, antioxidants, preservatives (e.g.antibacterial agents, antifungal agents), isotonic agents, absorptiondelaying agents, salts, preservatives, antioxidants, proteins, drugs,drug stabilizers, polymers, gels, binders, excipients, disintegrationagents, lubricants, sweetening agents, flavoring agents, dyes, such likematerials and combinations thereof, as would be known to one of ordinaryskill in the art (see, for example, Remington's Pharmaceutical Sciences,18th Ed. Mack Printing Company, 1990, pp. 1289-1329, incorporated hereinby reference). Except insofar as any conventional carrier isincompatible with the active ingredient, its use in the therapeutic orpharmaceutical compositions is contemplated.

An antibody or bispecific antigen binding molecule of the invention (andany additional therapeutic agent) can be administered by any suitablemeans, including parenteral, intrapulmonary, and intranasal, and, ifdesired for local treatment, intralesional administration. Parenteralinfusions include intramuscular, intravenous, intraarterial,intraperitoneal, or subcutaneous administration. Dosing can be by anysuitable route, e.g. by injections, such as intravenous or subcutaneousinjections, depending in part on whether the administration is brief orchronic.

Parenteral compositions include those designed for administration byinjection, e.g. subcutaneous, intradermal, intralesional, intravenous,intraarterial intramuscular, intrathecal or intraperitoneal injection.For injection, the antibodies or bispecific antigen binding molecules ofthe invention may be formulated in aqueous solutions, preferably inphysiologically compatible buffers such as Hanks' solution, Ringer'ssolution, or physiological saline buffer. The solution may containformulatory agents such as suspending, stabilizing and/or dispersingagents. Alternatively, the antibodies or bispecific antigen bindingmolecules may be in powder form for constitution with a suitablevehicle, e.g., sterile pyrogen-free water, before use. Sterileinjectable solutions are prepared by incorporating the antibodies orbispecific antigen binding molecules of the invention in the requiredamount in the appropriate solvent with various of the other ingredientsenumerated below, as required. Sterility may be readily accomplished,e.g., by filtration through sterile filtration membranes. Generally,dispersions are prepared by incorporating the various sterilized activeingredients into a sterile vehicle which contains the basic dispersionmedium and/or the other ingredients. In the case of sterile powders forthe preparation of sterile injectable solutions, suspensions oremulsion, the preferred methods of preparation are vacuum-drying orfreeze-drying techniques which yield a powder of the active ingredientplus any additional desired ingredient from a previouslysterile-filtered liquid medium thereof. The liquid medium should besuitably buffered if necessary and the liquid diluent first renderedisotonic prior to injection with sufficient saline or glucose. Thecomposition must be stable under the conditions of manufacture andstorage, and preserved against the contaminating action ofmicroorganisms, such as bacteria and fungi. It will be appreciated thatendotoxin contamination should be kept minimally at a safe level, forexample, less that 0.5 ng/mg protein. Suitable pharmaceuticallyacceptable carriers include, but are not limited to: buffers such asphosphate, citrate, and other organic acids; antioxidants includingascorbic acid and methionine; preservatives (such asoctadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride; benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecularweight (less than about 10 residues) polypeptides; proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, histidine, arginine, or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrins; chelating agents such as EDTA; sugars such as sucrose,mannitol, trehalose or sorbitol; salt-forming counter-ions such assodium; metal complexes (e.g. Zn-protein complexes); and/or non-ionicsurfactants such as polyethylene glycol (PEG). Aqueous injectionsuspensions may contain compounds which increase the viscosity of thesuspension, such as sodium carboxymethyl cellulose, sorbitol, dextran,or the like. Optionally, the suspension may also contain suitablestabilizers or agents which increase the solubility of the compounds toallow for the preparation of highly concentrated solutions.Additionally, suspensions of the active compounds may be prepared asappropriate oily injection suspensions. Suitable lipophilic solvents orvehicles include fatty oils such as sesame oil, or synthetic fatty acidesters, such as ethyl cleats or triglycerides, or liposomes.

Active ingredients may be entrapped in microcapsules prepared, forexample, by coacervation techniques or by interfacial polymerization,for example, hydroxymethylcellulose or gelatin-microcapsules andpoly-(methylmethacylate) microcapsules, respectively, in colloidal drugdelivery systems (for example, liposomes, albumin microspheres,microemulsions, nano-particles and nanocapsules) or in macroemulsions.Such techniques are disclosed in Remington's Pharmaceutical Sciences(18th Ed. Mack Printing Company, 1990). Sustained-release preparationsmay be prepared. Suitable examples of sustained-release preparationsinclude semipermeable matrices of solid hydrophobic polymers containingthe polypeptide, which matrices are in the form of shaped articles, e.g.films, or microcapsules. In particular embodiments, prolonged absorptionof an injectable composition can be brought about by the use in thecompositions of agents delaying absorption, such as, for example,aluminum monostearate, gelatin or combinations thereof.

In addition to the compositions described previously, the antibodies orbispecific antigen binding molecules may also be formulated as a depotpreparation. Such long acting formulations may be administered byimplantation (for example subcutaneously or intramuscularly) or byintramuscular injection. Thus, for example, the antibodies or bispecificantigen binding molecules may be formulated with suitable polymeric orhydrophobic materials (for example as an emulsion in an acceptable oil)or ion exchange resins, or as sparingly soluble derivatives, forexample, as a sparingly soluble salt.

Pharmaceutical compositions comprising the antibodies or bispecificantigen binding molecules of the invention may be manufactured by meansof conventional mixing, dissolving, emulsifying, encapsulating,entrapping or lyophilizing processes. Pharmaceutical compositions may beformulated in conventional manner using one or more physiologicallyacceptable carriers, diluents, excipients or auxiliaries whichfacilitate processing of the proteins into preparations that can be usedpharmaceutically. Proper formulation is dependent upon the route ofadministration chosen.

The antibodies or bispecific antigen binding molecules may be formulatedinto a composition in a free acid or base, neutral or salt form.Pharmaceutically acceptable salts are salts that substantially retainthe biological activity of the free acid or base. These include the acidaddition salts, e.g., those formed with the free amino groups of aproteinaceous composition, or which are formed with inorganic acids suchas for example, hydrochloric or phosphoric acids, or such organic acidsas acetic, oxalic, tartaric or mandelic acid. Salts formed with the freecarboxyl groups can also be derived from inorganic bases such as forexample, sodium, potassium, ammonium, calcium or ferric hydroxides; orsuch organic bases as isopropylamine, trimethylamine, histidine orprocaine. Pharmaceutical salts tend to be more soluble in aqueous andother protic solvents than are the corresponding free base forms.

Therapeutic Methods and Compositions

Any of the antibodies or bispecific antigen binding molecules providedherein may be used in therapeutic methods. Antibodies or bispecificantigen binding molecules of the invention may be used asimmunotherapeutic agents, for example in the treatment of cancers.

For use in therapeutic methods, antibodies or bispecific antigen bindingmolecules of the invention would be formulated, dosed, and administeredin a fashion consistent with good medical practice. Factors forconsideration in this context include the particular disorder beingtreated, the particular mammal being treated, the clinical condition ofthe individual patient, the cause of the disorder, the site of deliveryof the agent, the method of administration, the scheduling ofadministration, and other factors known to medical practitioners.

In one aspect, antibodies or bispecific antigen binding molecules of theinvention for use as a medicament are provided. In further aspects,antibodies or bispecific antigen binding molecules of the invention foruse in treating a disease are provided. In certain embodiments,antibodies or bispecific antigen binding molecules of the invention foruse in a method of treatment are provided.

In one embodiment, the invention provides an antibody or bispecificantigen binding molecule as described herein for use in the treatment ofa disease in an individual in need thereof. In certain embodiments, theinvention provides an antibody or bispecific antigen binding moleculefor use in a method of treating an individual having a diseasecomprising administering to the individual a therapeutically effectiveamount of the antibody or bispecific antigen binding molecule. Incertain embodiments the disease to be treated is a proliferativedisorder. In a particular embodiment the disease is cancer. In certainembodiments the method further comprises administering to the individuala therapeutically effective amount of at least one additionaltherapeutic agent, e.g., an anti-cancer agent if the disease to betreated is cancer. In further embodiments, the invention provides anantibody or bispecific antigen binding molecule as described herein foruse in inducing lysis of a target cell, particularly a tumor cell. Incertain embodiments, the invention provides an antibody or bispecificantigen binding molecule for use in a method of inducing lysis of atarget cell, particularly a tumor cell, in an individual comprisingadministering to the individual an effective amount of the antibody orbispecific antigen binding molecule to induce lysis of a target cell. An“individual” according to any of the above embodiments is a mammal,preferably a human.

In a further aspect, the invention provides for the use of an antibodyor bispecific antigen binding molecule of the invention in themanufacture or preparation of a medicament. In one embodiment themedicament is for the treatment of a disease in an individual in needthereof. In a further embodiment, the medicament is for use in a methodof treating a disease comprising administering to an individual havingthe disease a therapeutically effective amount of the medicament. Incertain embodiments the disease to be treated is a proliferativedisorder. In a particular embodiment the disease is cancer. In oneembodiment, the method further comprises administering to the individuala therapeutically effective amount of at least one additionaltherapeutic agent, e.g., an anti-cancer agent if the disease to betreated is cancer. In a further embodiment, the medicament is forinducing lysis of a target cell, particularly a tumor cell. In still afurther embodiment, the medicament is for use in a method of inducinglysis of a target cell, particularly a tumor cell, in an individualcomprising administering to the individual an effective amount of themedicament to induce lysis of a target cell. An “individual” accordingto any of the above embodiments may be a mammal, preferably a human.

In a further aspect, the invention provides a method for treating adisease. In one embodiment, the method comprises administering to anindividual having such disease a therapeutically effective amount of anantibody or bispecific antigen binding molecule of the invention. In oneembodiment a composition is administered to said individual, comprisingthe antibody or bispecific antigen binding molecule of the invention ina pharmaceutically acceptable form. In certain embodiments the diseaseto be treated is a proliferative disorder. In a particular embodimentthe disease is cancer.

In certain embodiments the method further comprises administering to theindividual a therapeutically effective amount of at least one additionaltherapeutic agent, e.g., an anti-cancer agent if the disease to betreated is cancer. An “individual” according to any of the aboveembodiments may be a mammal, preferably a human.

In a further aspect, the invention provides a method for inducing lysisof a target cell, particularly a tumor cell. In one embodiment themethod comprises contacting a target cell with an antibody or bispecificantigen binding molecule of the invention in the presence of a T cell,particularly a cytotoxic T cell. In a further aspect, a method forinducing lysis of a target cell, particularly a tumor cell, in anindividual is provided. In one such embodiment, the method comprisesadministering to the individual an effective amount of an antibody orbispecific antigen binding molecule to induce lysis of a target cell. Inone embodiment, an “individual” is a human.

In certain embodiments the disease to be treated is a proliferativedisorder, particularly cancer. Non-limiting examples of cancers includehaematological cancer such as leukemia, bladder cancer, brain cancer,head and neck cancer, pancreatic cancer, biliary cancer, thyroid cancer,lung cancer, breast cancer, ovarian cancer, uterine cancer, cervicalcancer, endometrial cancer, esophageal cancer, colon cancer, colorectalcancer, rectal cancer, gastric cancer, prostate cancer, skin cancer,squamous cell carcinoma, sarcoma, bone cancer, and kidney cancer. Othercell proliferation disorders that may be treated using an antibody orbispecific antigen binding molecule of the present invention include,but are not limited to neoplasms located in the: abdomen, bone, breast,digestive system, liver, pancreas, peritoneum, endocrine glands(adrenal, parathyroid, pituitary, testicles, ovary, thymus, thyroid),eye, head and neck, nervous system (central and peripheral), lymphaticsystem, pelvic, skin, soft tissue, spleen, thoracic region, andurogenital system. Also included are pre-cancerous conditions or lesionsand cancer metastases.

In certain embodiments the cancer is chosen from the group consisting ofhaematological cancer (such as leukemia), kidney cancer, bladder cancer,skin cancer, lung cancer, colorectal cancer, breast cancer, braincancer, head and neck cancer and prostate cancer. In one embodiment, thecancer is a haematological cancer, particularly leukemia, mostparticularly acute lymphoblastic leukemia (ALL) or acute myelogenousleukemia (AML). A skilled artisan readily recognizes that in many casesthe antibody or bispecific antigen binding molecule may not provide acure but may only provide partial benefit. In some embodiments, aphysiological change having some benefit is also consideredtherapeutically beneficial. Thus, in some embodiments, an amount ofantibody or bispecific antigen binding molecule that provides aphysiological change is considered an “effective amount” or a“therapeutically effective amount”. The subject, patient, or individualin need of treatment is typically a mammal, more specifically a human.

In some embodiments, an effective amount of an antibody or bispecificantigen binding molecule of the invention is administered to a cell. Inother embodiments, a therapeutically effective amount of an antibody orbispecific antigen binding molecule of the invention is administered toan individual for the treatment of disease.

For the prevention or treatment of disease, the appropriate dosage of anantibody or bispecific antigen binding molecule of the invention (whenused alone or in combination with one or more other additionaltherapeutic agents) will depend on the type of disease to be treated,the route of administration, the body weight of the patient, the type ofantibody or bispecific antigen binding molecule, the severity and courseof the disease, whether the antibody or bispecific antigen bindingmolecule is administered for preventive or therapeutic purposes,previous or concurrent therapeutic interventions, the patient's clinicalhistory and response to the antibody or bispecific antigen bindingmolecule, and the discretion of the attending physician. Thepractitioner responsible for administration will, in any event,determine the concentration of active ingredient(s) in a composition andappropriate dose(s) for the individual subject. Various dosing schedulesincluding but not limited to single or multiple administrations overvarious time-points, bolus administration, and pulse infusion arecontemplated herein.

The antibody or bispecific antigen binding molecule is suitablyadministered to the patient at one time or over a series of treatments.Depending on the type and severity of the disease, about 1 ng/kg to 15mg/kg (e.g. 0.1 mg/kg-10 mg/kg) of antibody or bispecific antigenbinding molecule can be an initial candidate dosage for administrationto the patient, whether, for example, by one or more separateadministrations, or by continuous infusion. One typical daily dosagemight range from about 1 μg/kg to 100 mg/kg or more, depending on thefactors mentioned above. For repeated administrations over several daysor longer, depending on the condition, the treatment would generally besustained until a desired suppression of disease symptoms occurs. Oneexemplary dosage of the antibody or bispecific antigen binding moleculewould be in the range from about 0.005 mg/kg to about 10 mg/kg. In othernon-limiting examples, a dose may also comprise from about 1microgram/kg body weight, about 5 microgram/kg body weight, about 10microgram/kg body weight, about 50 microgram/kg body weight, about 100microgram/kg body weight, about 200 microgram/kg body weight, about 350microgram/kg body weight, about 500 microgram/kg body weight, about 1milligram/kg body weight, about 5 milligram/kg body weight, about 10milligram/kg body weight, about 50 milligram/kg body weight, about 100milligram/kg body weight, about 200 milligram/kg body weight, about 350milligram/kg body weight, about 500 milligram/kg body weight, to about1000 mg/kg body weight or more per administration, and any rangederivable therein. In non-limiting examples of a derivable range fromthe numbers listed herein, a range of about 5 mg/kg body weight to about100 mg/kg body weight, about 5 microgram/kg body weight to about 500milligram/kg body weight, etc., can be administered, based on thenumbers described above. Thus, one or more doses of about 0.5 mg/kg, 2.0mg/kg, 5.0 mg/kg or 10 mg/kg (or any combination thereof) may beadministered to the patient. Such doses may be administeredintermittently, e.g. every week or every three weeks (e.g. such that thepatient receives from about two to about twenty, or e.g. about six dosesof the antibody or bispecific antigen binding molecule). An initialhigher loading dose, followed by one or more lower doses may beadministered. However, other dosage regimens may be useful. The progressof this therapy is easily monitored by conventional techniques andassays.

The antibodies or bispecific antigen binding molecules of the inventionwill generally be used in an amount effective to achieve the intendedpurpose. For use to treat or prevent a disease condition, the antibodiesor bispecific antigen binding molecules of the invention, orpharmaceutical compositions thereof, are administered or applied in atherapeutically effective amount. Determination of a therapeuticallyeffective amount is well within the capabilities of those skilled in theart, especially in light of the detailed disclosure provided herein.

For systemic administration, a therapeutically effective dose can beestimated initially from in vitro assays, such as cell culture assays. Adose can then be formulated in animal models to achieve a circulatingconcentration range that includes the IC₅₀ as determined in cellculture. Such information can be used to more accurately determineuseful doses in humans.

Initial dosages can also be estimated from in vivo data, e.g., animalmodels, using techniques that are well known in the art. One havingordinary skill in the art could readily optimize administration tohumans based on animal data.

Dosage amount and interval may be adjusted individually to provideplasma levels of the antibodies or bispecific antigen binding moleculeswhich are sufficient to maintain therapeutic effect. Usual patientdosages for administration by injection range from about 0.1 to 50mg/kg/day, typically from about 0.5 to 1 mg/kg/day. Therapeuticallyeffective plasma levels may be achieved by administering multiple doseseach day. Levels in plasma may be measured, for example, by HPLC.

In cases of local administration or selective uptake, the effectivelocal concentration of the antibodies or bispecific antigen bindingmolecules may not be related to plasma concentration. One having skillin the art will be able to optimize therapeutically effective localdosages without undue experimentation.

A therapeutically effective dose of the antibodies or bispecific antigenbinding molecules described herein will generally provide therapeuticbenefit without causing substantial toxicity. Toxicity and therapeuticefficacy of an antibody or bispecific antigen binding molecule can bedetermined by standard pharmaceutical procedures in cell culture orexperimental animals. Cell culture assays and animal studies can be usedto determine the LD₅₀ (the dose lethal to 50% of a population) and theED₅₀ (the dose therapeutically effective in 50% of a population). Thedose ratio between toxic and therapeutic effects is the therapeuticindex, which can be expressed as the ratio LD₅₀/ED₅₀. Antibodies orbispecific antigen binding molecules that exhibit large therapeuticindices are preferred. In one embodiment, the antibody or bispecificantigen binding molecule according to the present invention exhibits ahigh therapeutic index. The data obtained from cell culture assays andanimal studies can be used in formulating a range of dosages suitablefor use in humans. The dosage lies preferably within a range ofcirculating concentrations that include the ED₅₀ with little or notoxicity. The dosage may vary within this range depending upon a varietyof factors, e.g., the dosage form employed, the route of administrationutilized, the condition of the subject, and the like. The exactformulation, route of administration and dosage can be chosen by theindividual physician in view of the patient's condition (see, e.g.,Fingl et al., 1975, in: The Pharmacological Basis of Therapeutics, Ch.1, p. 1, incorporated herein by reference in its entirety).

The attending physician for patients treated with antibodies orbispecific antigen binding molecules of the invention would know how andwhen to terminate, interrupt, or adjust administration due to toxicity,organ dysfunction, and the like. Conversely, the attending physicianwould also know to adjust treatment to higher levels if the clinicalresponse were not adequate (precluding toxicity). The magnitude of anadministered dose in the management of the disorder of interest willvary with the severity of the condition to be treated, with the route ofadministration, and the like. The severity of the condition may, forexample, be evaluated, in part, by standard prognostic evaluationmethods. Further, the dose and perhaps dose frequency will also varyaccording to the age, body weight, and response of the individualpatient.

Other Agents and Treatments

The antibodies and bispecific antigen binding molecules of the inventionmay be administered in combination with one or more other agents intherapy. For instance, an antibody or bispecific antigen bindingmolecule of the invention may be co-administered with at least oneadditional therapeutic agent. The term “therapeutic agent” encompassesany agent administered to treat a symptom or disease in an individual inneed of such treatment. Such additional therapeutic agent may compriseany active ingredients suitable for the particular indication beingtreated, preferably those with complementary activities that do notadversely affect each other. In certain embodiments, an additionaltherapeutic agent is an immunomodulatory agent, a cytostatic agent, aninhibitor of cell adhesion, a cytotoxic agent, an activator of cellapoptosis, or an agent that increases the sensitivity of cells toapoptotic inducers. In a particular embodiment, the additionaltherapeutic agent is an anti-cancer agent, for example a microtubuledisruptor, an antimetabolite, a topoisomerase inhibitor, a DNAintercalator, an alkylating agent, a hormonal therapy, a kinaseinhibitor, a receptor antagonist, an activator of tumor cell apoptosis,or an antiangiogenic agent.

Such other agents are suitably present in combination in amounts thatare effective for the purpose intended. The effective amount of suchother agents depends on the amount of antibody or bispecific antigenbinding molecule used, the type of disorder or treatment, and otherfactors discussed above. The antibodies or bispecific antigen bindingmolecules are generally used in the same dosages and with administrationroutes as described herein, or about from 1 to 99% of the dosagesdescribed herein, or in any dosage and by any route that isempirically/clinically determined to be appropriate.

Such combination therapies noted above encompass combined administration(where two or more therapeutic agents are included in the same orseparate compositions), and separate administration, in which case,administration of the antibody or bispecific antigen binding molecule ofthe invention can occur prior to, simultaneously, and/or following,administration of the additional therapeutic agent and/or adjuvant.Antibodies or bispecific antigen binding molecules of the invention mayalso be used in combination with radiation therapy.

Articles of Manufacture

In another aspect of the invention, an article of manufacture containingmaterials useful for the treatment, prevention and/or diagnosis of thedisorders described above is provided. The article of manufacturecomprises a container and a label or package insert on or associatedwith the container. Suitable containers include, for example, bottles,vials, syringes, IV solution bags, etc. The containers may be formedfrom a variety of materials such as glass or plastic. The containerholds a composition which is by itself or combined with anothercomposition effective for treating, preventing and/or diagnosing thecondition and may have a sterile access port (for example the containermay be an intravenous solution bag or a vial having a stopper pierceableby a hypodermic injection needle). At least one active agent in thecomposition is an antibody or bispecific antigen binding molecule of theinvention. The label or package insert indicates that the composition isused for treating the condition of choice. Moreover, the article ofmanufacture may comprise (a) a first container with a compositioncontained therein, wherein the composition comprises an antibody orbispecific antigen binding molecule of the invention; and (b) a secondcontainer with a composition contained therein, wherein the compositioncomprises a further cytotoxic or otherwise therapeutic agent. Thearticle of manufacture in this embodiment of the invention may furthercomprise a package insert indicating that the compositions can be usedto treat a particular condition. Alternatively, or additionally, thearticle of manufacture may further comprise a second (or third)container comprising a pharmaceutically-acceptable buffer, such asbacteriostatic water for injection (BWFI), phosphate-buffered saline,Ringer's solution and dextrose solution. It may further include othermaterials desirable from a commercial and user standpoint, includingother buffers, diluents, filters, needles, and syringes.

Methods and Compositions for Diagnostics and Detection

In certain embodiments, any of the anti-HLA-A2/WT1 antibodies providedherein is useful for detecting the presence of HLA-A2/WT1 in abiological sample. The term “detecting” as used herein encompassesquantitative or qualitative detection. In certain embodiments, abiological sample comprises a cell or tissue, such as prostate tissue.

In one embodiment, an anti-HLA-A2/WT1 antibody for use in a method ofdiagnosis or detection is provided. In a further aspect, a method ofdetecting the presence of HLA-A2/WT1 in a biological sample is provided.In certain embodiments, the method comprises contacting the biologicalsample with an anti-HLA-A2/WT1 antibody as described herein underconditions permissive for binding of the anti-HLA-A2/WT1 antibody toHLA-A2/WT1, and detecting whether a complex is formed between theanti-HLA-A2/WT1 antibody and HLA-A2/WT1. Such method may be an in vitroor in vivo method. In one embodiment, an anti-HLA-A2/WT1 antibody isused to select subjects eligible for therapy with an anti-HLA-A2/WT1antibody, e.g. where HLA-A2/WT1 is a biomarker for selection ofpatients.

Exemplary disorders that may be diagnosed using an antibody of theinvention include cancer, particularly prostate cancer.

In certain embodiments, labeled anti-HLA-A2/WT1 antibodies are provided.Labels include, but are not limited to, labels or moieties that aredetected directly (such as fluorescent, chromophoric, electron-dense,chemiluminescent, and radioactive labels), as well as moieties, such asenzymes or ligands, that are detected indirectly, e.g., through anenzymatic reaction or molecular interaction. Exemplary labels include,but are not limited to, the radioisotopes ³²P, ¹⁴C, ¹²⁵I, ³H, and ¹³¹I,fluorophores such as rare earth chelates or fluorescein and itsderivatives, rhodamine and its derivatives, dansyl, umbelliferone,luceriferases, e.g., firefly luciferase and bacterial luciferase (U.S.Pat. No. 4,737,456), luciferin, 2,3-dihydrophthalazinediones,horseradish peroxidase (HRP), alkaline phosphatase, β-galactosidase,glucoamylase, lysozyme, saccharide oxidases, e.g., glucose oxidase,galactose oxidase, and glucose-6-phosphate dehydrogenase, heterocyclicoxidases such as uricase and xanthine oxidase, coupled with an enzymethat employs hydrogen peroxide to oxidize a dye precursor such as HRP,lactoperoxidase, or microperoxidase, biotin/avidin, spin labels,bacteriophage labels, stable free radicals, and the like.

EXAMPLES

The following are examples of methods and compositions of the invention.It is understood that various other embodiments may be practiced, giventhe general description provided above.

Example 1. Generation of Fab Binders to HLA-A2/WT1 Selection andScreening of Anti-HLA-A2/WT1 Fabs

Anti-HLA-A2/WT1 Fabs were selected by phage display from synthetic Fablibraries based on entirely human frameworks with sequence diversity inCDR3 of VL (3 different lengths) and VH domains (6 different lengths).

Selection rounds (biopanning) were performed in solution according tothe following protocol: 1. pre-clearing of ˜10¹² phagemid particles perlibrary pool on neutravidin coated 96 well plates coated with 500 nM ofan unrelated biotinylated HLA-A2/WT1 μm complex, 2. incubation of thenon-HLA-A2/WT1_(VLD)-binding phagemid particles with 100 nM biotinylatedHLA-A2/WT1_(RMF) complex for 0.5 h in a total volume of 800 μl, 3.capture of biotinylated HLA-A2/WT1_(RMF) and specifically binding phageby adding 80 μl of streptavidin-coated magnetic particles for 20 min ona shaker, 4. washing of respective magnetic particles 5-10× with 1 mlPBS/Tween 20 and 5-10× with 1 ml PBS using a magnetic particleseparator, 5. elution of phage particles by addition of 1 ml 100 mMtriethylamine (TEA) for 5-10 min and neutralization by addition of an ½volume of 1 M Tris/HCl pH 7.4, 6. re-infection of log-phase E. coli TG1cells with the eluted phage particles, infection with helperphageVCSM13, incubation on a shaker at 30° C. over night and subsequentPEG/NaCl precipitation of phagemid particles to be used in the nextselection round.

Selections were carried out over 3 to 4 rounds using constant antigenconcentrations of 100 nM.

In addition to selection campaigns with constant antigen concentrations,further selection campaigns were carried out with decreasing antigenconcentrations of 100 nM, 50 nM, 10 nM and 5 nM in order to select forantibodies with lower affinities.

HLA-A2/WT1 Binding Assays: Sandwich ELISA for Characterisation of FabsObtained by Phage Display

Individual clones were bacterially expressed as 1 ml cultures in 96-wellformat and supernatants were subjected to a screening by ELISA. Specificbinders were defined as having signals higher than 5× background forHLA-A2/WT1_(RMF) and signals lower than 3× background forHLA-A2/WT1_(VLD). More precisely, neutravidin 96 well strip plates(Thermo Fisher) were coated with 10 nM of HLA-A2/WT1_(RMF) or 50 nMHLA-A2/WT1_(VLD)) at 37° C. for 30 min, followed by blocking of theplate with 2% (w/v) milk-phosphate-buffered saline (MPBS) (200 μl/well)for 1 h at room temperature. The plate was washed 3 times with PBS, thenFab containing bacterial supernatants were added and the plate wasincubated at room temperature for 1 h. After another 3 washing stepswith PBS, anti-FLAG-HRP secondary antibody (Sigma, Cat. No. A8592)((1:4000) was added and the plate was incubated on a shaker for 1 h atroom temperature. The plate was washed 3 times with PBS and developed byadding 100 μl/well BM Blue POD (Roche). The enzymatic reaction wasstopped by adding 50 μl/well 1 M H2504. The OD was read at 450 nm(reference at 650 nm) for a final read-out of OD₄₅₀₋₆₅₀. ELISA-positiveclones were subjected to the kinetic screening experiment describedbelow.

HLA-A2/WT1 Binding Assays: Surface Plasmon Resonance for KineticCharacterisation of Fabs Obtained by Phage Display

Specific binders were identified by surface plasmon resonance-screeningof Fab-containing bacterial culture supernatants using a ProteOn XPR36biosensor (BioRad). In brief, after infection of log-phase E. coli TG1cells with the eluted phage particles, single colony forming units (cfu)were plated and picked for inoculation of 1 ml expression cultures in96-deep well plates.

All experiments were performed at 25° C. using PBST as running buffer(10 mM PBS, pH 7.4 and 0.005% (v/v) Tween 20). A ProteOn XPR36 biosensorequipped with GLC and GLM sensor chips and coupling reagents (10 mMsodium acetate pH 4.5, sulfo-N-hydroxysuccinimide [sulfo-NHS],1-ethyl-3-(3-dimethylaminpropyl)-carbodiimide hydrochloride [EDC] andethanolamine) from BioRad Inc. (Hercules, Calif.) were used.

Immobilizations were performed at 30 μl/min on a GLM chip. pAb (goat)anti human IgG, F(ab)₂ specific antibody (Jackson ImmunoResearch) wascoupled in vertical direction using a standard amine-coupling procedure:all six ligand channels were activated for 5 min with a mixture of EDC(200 mM) and sulfo-NHS (50 mM). Immediately after the surfaces wereactivated, pAb (goat) anti human IgG, F(ab)₂ specific antibody (50μg/ml, 10 mM sodium acetate, pH 5) was injected across all six channelsfor 5 min. Finally, channels were blocked with a 5 min injection of 1 Methanolamine-HCl (pH 8.5). Final immobilization levels were similar onall channels, ranging from 11000 to 11500 RU. The Fab variants werecaptured from E. coli supernatants by simultaneous injection along fiveof the separate horizontal channels (30 μl/min) for 5 min and resultedin levels ranging from 200 to 900 RU, depending on the concentration ofFab in supernatant. Conditioned medium was injected along the sixthchannel to provide an ‘in-line’ blank for double referencing purposes.One-shot kinetic measurements were performed by injection of a dilutionseries of HLA-A2/WT1_(RMF) or HLA-A2/WT1_(VLD) (100, 50, 25, 12.5, 6.25,0 nM, 50 μl/min) for 2 min along the vertical channels. Dissociation wasmonitored for 3 min. Kinetic data were analyzed in ProteOn Manager v.2.1. Processing of the reaction spot data involved applying aninterspot-reference and a double-reference step using an inline bufferblank (Myszka, J Mol Recognit (1999) 12, 279-284). The processed datafrom replicate one-shot injections were fit to a simple 1:1 Langmuirbinding model without mass transport (O'Shannessy et al., Anal Biochem(1993) 212, 457-468).

For measurements of IgG from supernatants of HEK productions in 6-wellformat, the IgG variants were captured from HEK293 supernatants bysimultaneous injection along five of the separate horizontal channels(30 μl/min) for 5 min and resulted in levels ranging from 200 to 400 RU.Conditioned medium was injected along the sixth channel to provide an‘in-line’ blank for double referencing purposes. One-shot kineticmeasurements were performed by injection of a dilution series ofHLA-A2/WT1_(RMF) or HLA-A2/WT1_(VLD) (100, 50, 25, 12.5, 6.25, 0 nM, 50μl/min) for 3 min along the vertical channels. Dissociation wasmonitored for 5 min. Kinetic data were analyzed as described above.

Based on binding profile and measured specificity to bind to theantigen, binders were shortlisted and measured in cell binding assays.

Sequences of all selected binders (11D06, 33H09, 13B04, 11B09, 33F05,5E11, 13E08, 5C01, 11G06) are provided in the sequence listing includedherein and summarized in Table 1 below.

TABLE 1 Amino acid sequences of selected HLA-A2/WT1 binders. SEQ ID NOBinder HCDR1 HCDR2 HCDR3 VH LCDR1 LCDR2 LCDR3 VL 11D06 1 2 3 7 4 5 6 833H09 9 10 11 15 12 13 14 16 13B04 17 18 19 23 20 21 22 24 11B09 25 2627 31 28 29 30 32 33F05 33 34 35 39 36 37 38 40 5E11 41 42 43 47 44 4546 48 13E08 49 50 51 55 52 53 54 56 5C01 57 58 59 63 60 61 62 64 11G0665 66 67 71 68 69 70 72

Example 2. Preparation of HLA-A2/WT1 IgG and HLA-A2/WT1×CD3 BispecificAntibodies Cloning

The cDNAs encoding the proteins were cloned into a vector system(Evitria) using conventional (non-PCR based) cloning techniques. Thevector plasmids were gene synthesized. Plasmid DNA was prepared underlow-endotoxin conditions based on anion exchange chromatography. DNAconcentration was determined by measuring the absorption at a wavelengthof 260 nm. Correctness of the sequences was verified with Sangersequencing (with up to two sequencing reactions per plasmid depending onthe size of the cDNA).

Production

IgG antibodies and bispecific antibodies were generated by transienttransfection of HEK293 EBNA cells (cultivated in suspension serum freein Excell culture medium). Cells were centrifuged and medium replaced bypre-warmed CD CHO medium. Expression vectors were mixed in CD CHOmedium, polyethyleneimine (PEI) was added, the solution vortexed andincubated for 10 minutes at room temperature. Afterwards, cells weremixed with the DNA/PEI solution, transferred to shake flask andincubated for 3 hours at 37° C. in an incubator with a 5% CO₂atmosphere. After the incubation, Excell medium with supplements wasadded. One day after transfection supplements were added. Cellsupernatants were harvested after 7 days and purified by standardmethods.

Alternatively, suspension-adapted CHO K1 cells (originally from ATCC andadapted to serum-free growth in suspension culture) were used forproduction. The seed was grown in eviGrow medium, a chemically defined,animal-component free, serum-free medium. Cells were transfected witheviFect transfection reagent, and cells were grown after transfection ineviMake2, an animal-component free, serum-free medium. Supernatant washarvested by centrifugation and subsequent filtration (0.2 μm filter).

Purification

Proteins were purified from filtered cell culture supernatants referringto standard protocols. In brief, Fc containing proteins were purifiedfrom cell culture supernatants by affinity chromatography using ProteinA (HiTrap ProteinA HP column, GE Healthcare). Elution was achieved at pH3.0 followed by immediate neutralization of the sample. The protein wasconcentrated and aggregated protein was separated from monomeric proteinby size exclusion chromatography (HiLoad Superdex 200 column, GEHealthcare) in 20 mM histidine, 140 mM sodium chloride, pH 6.0.

Analytics

The concentration of purified proteins was determined by measuring theoptical density (OD) at 280 nm, using the molar extinction coefficientcalculated on the basis of the amino acid sequence according to Pace etal. (Protein Science, 1995, 4, 2411-1423). Purity and molecular weightof the proteins were analyzed by CE-SDS in the presence and absence of areducing agent using a LabChipGXII system (Caliper Lifescience).Determination of the aggregate content was performed by HPLCchromatography using analytical size-exclusion column (TSKgel G3000 SWXL, Tosoh) equilibrated in a 25 mM K2HPO₄, 125 mM NaCl, 200 mML-arginine monohydrocloride, pH 6.7 running buffer at 25° C.

CD3 bispecific antibodies are also referred to herein a “T cellbispecific antibodies” or “TCBs”. A schematic illustration of thebispecific antibodies prepared in this example is given in FIG. 2.

Exemplary sequences of TCBs are given in SEQ ID NOs 123, 124, 125 and129 (11D06 TCB) and SEQ ID NOs 126, 127, 128 and 129 (33H09-TCB). OtherTCBs were constructed in an analogous manner, using the VH and VLsequences of the corresponding HLA-A2/WT1 binders.

As controls, a HLA-A2/WT1×CD3 bispecific antibody (TCB) based on abinder similar to antibody ESK1 (Dao et al., Sci Transl Med (2013) 5,176ra33; WO2012/135854)—referred to herein as “ESK1-TCB” (see SEQ ID NOs73 and 74 for the variable region sequences)—as well as an untargetedTCB (see SEQ ID NOs 75 and 76 for the variable region sequences) wereprepared.

All molecules were produced and purified following the same method. Thefinal quality was very good for all molecules with almost 100% monomercontent and 100% purity on CE-SDS.

Example 3. Biochemical Analysis of Affinity and Avidity ofHLA-A2/WT1×CD3 Bispecific Antibodies

For determination of affinity of HLA-A2/WT1×CD3 bispecific antibodies toHLA-A2/WT1_(RMF), surface plasmon resonance (SPR) experiments wereperformed at 25° C. on a Biacore T200 with HBS-EP as running buffer(0.01 M HEPES pH 7.4, 0.15 M NaCl, 0.005% Surfactant P20 (GEHealthcare)). Anti-human Fc specific antibody (GE Healthcare, Cat. No.BR-1008-39) was directly immobilized by amine coupling on a CMS chip (GEHealthcare). The bispecific constructs were captured for 30 s at 5 nM. Athree-fold dilution series of the HLA-A2/WT1_(RMF) complex in HBS-EP(1.03 to 250 nM) was passed over the ligand at 30 μl/min for 120 sec torecord the association phase. The dissociation phase was monitored for120 s and triggered by switching from the sample solution to HBS-EP. Thechip surface was regenerated after every cycle using an injection of 3MMgCl₂ at 10 μl/min for 30 sec. Bulk refractive index differences werecorrected for by subtracting the response obtained on the reference flowcell which contains the anti-human Fc antibody, but without bispecificconstruct captured on it. The affinity constants were derived from thekinetic rate constants by fitting to a 1:1 Langmuir binding using theBIAeval software (GE Healthcare).

For determination of avidity and specificity of the HLA-A2/WT1×CD3bispecific antibodies to HLA-A2/WT1_(RMF), SPR experiments were againperformed at 25° C. on a Biacore T200 with HBS-EP as running buffer(0.01 M HEPES pH 7.4, 0.15 M NaCl, 0.005% Surfactant P20 (GEHealthcare)). Anti-His antibody (Penta His, Qiagen Cat. No. 34660) wasdirectly immobilized by amine coupling on a CMS chip (GE Healthcare).The HLA-A2/WT1_(RMF) or HLA-A2/WT1_(VLD) was captured for 30 sec and 10μl/min at 5 or 10 nM (for the bispecific antibody or IgG measurement,respectively). A 3-fold dilution series of the bispecific constructs inHBS-EP (1.23 to 100 nM) were passed over the ligand at 30 μl/min for 120sec to record the association phase. The dissociation phase wasmonitored for 240 sec and triggered by switching from the samplesolution to HBS-EP. The chip surface was regenerated after every cycleusing an injection of 10 mM glycine pH 2 for 60 sec. Bulk refractiveindex differences were corrected for by subtracting the responseobtained on the reference flow cell (which contains the anti-Hisantibody, but without HLA-A2/WT1_(RMF) captured on it). Even though itis a 2:1 interaction (analyte is bivalent) the affinity constants werederived from the kinetic rate constants by fitting to a 1:1 Langmuirbinding using the BIAeval software (GE Healthcare). This results in anapparent K_(D) representing the avidity of the interaction.

The results of these experiments are summarized in Table 2 below. Bothtested HLA-A2/WT1 antibodies bind to HLA-A2/WT1_(RMF) with double-digitnanomolar (monovalent) affinity/three-digit picomolar (bivalent)affinity (avidity), and keep the same affinity/avidity when convertedfrom IgG to bispecific format. While the affinity of the two testedantibodies differs by about a factor two, avidity of both molecules isin the same range.

No binding of the tested HLA-A2/WT1 antibodies to HLA-A2/WT1 μm wasdetected (data not shown).

TABLE 2 Summary of affinity and avidity data as determined by SPR forselected HLA-A2/WT1 IgG and HLA-A2/WT1 × CD3 bispecific antibodies(“TCBs”) to HLA-A2/WT1_(RMF). Avidity ka (1/Ms) kd (1/s) ApparentAffinity average average KD (pM) Analyte ligand ka (1/Ms) kd (1/s) KDstdev stdev stdev 33H09-huIgG1 RMF 1.67 10⁶ 1.16 10⁻¹ 70 nM 4.74 10⁶3.57 10⁻³ 750  1.8 10⁶ 1.24 10⁴   69 nM  6.5 10⁴ 2.35 10⁻⁴ 50 33H09-TCBRMF 2.67 10⁶ 1.85 10⁻¹ 70 nM    3.3 10⁻⁷ 1.81 10⁻² 540 2.32 10⁶ 1.6110⁻¹ 69 nM 5.85 10⁶ 6.45 10⁻³ 110 11D06-huIgG1 RMF 1.08 10⁶ 3.78 10⁻² 35nM 2.97 10⁶ 1.81 10⁻³ 610 1.04 10⁶ 3.57 10⁻² 34 nM 1.01 10⁵ 1.28 10⁻⁴ 2011D06-TCB RMF 1.07 10⁶ 3.85 10⁻² 36 nM 7.05 10⁶ 4.98 10⁻³ 710 1.12 10⁶3.82 10⁻² 34 nM 1.87 10⁶ 1.39 10⁻³ 80

For determination of affinity of HLA-A2/WT1×CD3 bispecific antibodies toCD3, surface plasmon resonance (SPR) experiments were performed at 25°C. on a Biacore T200 with HBS-EP as running buffer (0.01 M HEPES pH 7.4,0.15 M NaCl, 0.005% Surfactant P20 (GE Healthcare)). Anti-human Fabspecific antibody (GE Healthcare, Cat. No. 28-9583-25) was directlyimmobilized by amine coupling on a CMS chip (GE Healthcare). Thebispecific constructs were captured for 40 s at 5 nM. A three-folddilution series of the CD3εδ-Fc fusion molecule in HBS-EP (12.35 to 3000nM) was passed over the bispecific antibodies at 30 μl/min for 240 secto record the association phase. The dissociation phase was monitoredfor 240 s and triggered by switching from the sample solution to HBS-EP.The chip surface was regenerated after every cycle using a doubleinjection of 10 mM glycine-HCl pH 2.1 at 30 μl/min for 60 sec. Bulkrefractive index differences were corrected for by subtracting theresponse obtained on the reference flow cell (which contains theanti-human Fab antibody, but without bispecific construct captured onit). The affinity constants were derived from the kinetic rate constantsby fitting to a 1:1 Langmuir binding using the BIAeval software (GEHealthcare).

The results are summarized in Table 3. Since the CD3 binder is identicalin both tested bispecific molecule, as expected their affinity to CD3 isessentially the same.

TABLE 3 Summary of affinity data as determined by SPR for selectedHLA-A2/WT1 × CD3 bispecific antibodies (“TCBs”) to CD3. Affinity Analyteligand ka (1/Ms) kd (1/s) KD 33H09-TCB huCD3-Fc 2.74 10⁴ 2.93 10⁻³ 110nM 2.66 10⁴ 2.77 10⁻³ 100 nM 11D06-TCB huCD3-Fc 2.59 10⁴ 3.01 10⁻³ 120nM 2.53 10⁴ 2.82 10⁻³ 110 nM

Example 4. Selection of IgG Binders for Specificity for HLA-A2/WT1Peptide RMF or VLD

We first measured the binding specificity on peptide-pulsed T2 cells bythe IgG binders generated from phage display by flow cytometry. Briefly,T2 cells were prepared as a cell suspension at 10⁶ cells/ml in IMDMmedium (Gibco by Life Technologies, Cat No. 31980-048), supplementedwith 10% FBS (Gibco, Cat No. 16140-071)+1% Penicillin-Streptomycin(Gibco, Cat No. 15070-063) (complete medium). Cells were kept in a totalvolume of 10 ml in a tube, and incubated with 100 of peptide (WT1 p37-45VLD peptide (SEQ ID NO: 77), or p126-134 RMF peptide (SEQ ID NO: 78)) at10⁻² M (final concentration of the peptide: 10⁻⁵M) for 2 hours at 37° C.with 5% CO₂. After washing, cells were suspended in cold PBS andincubated with titrated concentration of IgG binders (10 μg/ml to0.00064 μg/ml) for 1 hour at 4° C., followed by incubation with asecondary anti-human IgG-Fc phycoerythrin (PE)-conjugated antibody(Jackson Laboratories, Cat No. 109-116-098) for 30 min. Cells wereacquired on FACS LSR II (BD), and data are presented as meanfluorescence intensity (MFI) of PE in Graphpad Prism.

As shown in FIG. 3, 11D06-IgG, 33H09-IgG and 5E11-IgG all bind toRMF-peptide pulsed T2 cells, but not to unpulsed or VLD peptide-pulsedT2 cells. In contrast, 11B09-IgG, 13B04-IgG and 5C01-IgG (but not11G06-IgG) all bind specifically to VLD-peptide, but not to RMF-peptide.Based on these data binders for conversion to CD3 bispecific antibodies(TCBs) were selected.

Example 5. Activation of T Cells in a NFAT-Jurkat Reporter Assay UponBinding of HLA-A2/WT1×CD3 Bispecific Antibodies (“TCBs”) toPeptide-Pulsed T2 Cells

To check the specificity of the HLA-A2/WT1×CD3 bispecific antibodies(“TCBs”), a reporter cell line, Jurkat cells that express luciferaseunder the promoter of NFAT (Jurkat-NFAT; Promega Cat. No. CS176501), wasused to measure activation of T cells when TCBs bind to peptide-pulsedT2 cells (ATCC, Cat. No. CRL-1992). Briefly, T2 cells were prepared as acell suspension at 10⁶ cells/ml in IMDM medium (Gibco by LifeTechnologies, Cat. No. 31980-048), supplemented with 10% FBS (Gibco,Cat. No. 16140-071)+1% Penicillin-Streptomycin (Gibco, Cat. No.15070-063) (complete medium). Cells were kept in a total volume of 10 mlin a tube, and incubated with 10⁶ of peptide (WT1 p37-45 VLD peptide, orp126-134 RMF peptide) at 10⁻²M (final concentration of the peptide:10⁻⁵M) for 2 hours at 37° C. with 5% CO₂. After washing, 90 μl of thepeptide-pulsed cells in a cell suspension of 2.2×10⁵ cells/ml wereseeded into a 96 well microtiter round bottom plate (20,000 cells/well,TPP, Cat. No. 92097), co-cultured with 50 μl of Jurkat-NFAT (cellsuspension of 2×10⁶ cells/ml), and with 10 μl of titrated TCB (at 100μg/ml to 0.0064 μg/ml in PBS) for 16 hours at 37° C. with 5% CO₂.Thereafter, 50 μl of supernatant were removed, and replaced with 100 μlper well of Bright-Glo Luciferase Assay (Promega, Cat. No. E2620) forincubation at room temperature (RT). Five minutes later, 180 μl ofsupernatant were transferred into a new white plate to measureluminescence signal by EnVision (PerkinElmer). Data are presented asRelative Luminescence Unit (RLU).

As shown in FIG. 4, the TCBs based on the 11D06 and the 33H09 binder(11D06-TCB and 33H09-TCB, respectively) specifically recognize theHLA-A2/WT1_(RMF) complex and activate NFAT on reporter Jurkat cells onlyin the presence of T2 cells pulsed with the RMF peptide. In contrastthereto, the control TCB based on the ESK1-like binder (ESK1-TCB) didnot show specificity for RMF peptide. Also the TCB based on the 5E11binder (5E11-TCB) showed activation of NFAT reporter T cells in thepresence of both RMF and VLD peptide-pulsed T2 cells, thus this TCB waseliminated for the next round of screening. There were also some VLDpeptide-specific TCBs identified such as the ones based on the 11B09 andthe 13B04 binder (11B09-TCB and 13B04-TCB, respectively). As a negativecontrol, untargeted TCB (DP47GS-TCB) was used in the assay, which didnot activate NFAT on reporter Jurkat cells in the presence of RMF- orVLD-peptide pulsed T2 cells. 5C01-TCB showed recognition of VLD peptide,but cross-reacted with RMF peptide at higher concentrations.

Example 6. T Cell Cytotoxicity Mediated by HLA-A2/WT1×CD3 BispecificAntibodies (“TCBs”) Upon Binding to Peptide-Pulsed T2 Cells

Next, we measured the cytotoxicity of the TCBs. The target cells werepeptide-pulsed T2 cells as described in Example 5. The effector cellswere pan CD3⁺ cells purified from PBMCs isolated from buffy coat byFicoll (GE Healthcare, Cat. No. 17-1440-03) gradient centrifugation.Total CD3⁺ T cells were purified by MACS (Miltenyi Biotec) using a HumanPan T cell Isolation Kit (Miltenyi Biotec, Cat. No. 130-096-535). Thecytotoxicity assay was performed as follows: The peptide-pulsed cells(100 μl) were seeded into a 96 well microtiter round bottom plate (3×10⁵cells/ml), co-cultured with 50 μl of T cells (6×10⁶ cells/nil), and with50 μl of titrated TCB (at 40 μg/ml to 0.00004 μg/ml) in complete mediumfor 18 hours at 37° C. with 5% CO₂. Thereafter, 50 μl of supernatantwere transferred into a new white plate, and 25 μl per well ofCytoTox-Glo Luciferase Assay (Promega, Cat. No. G9291) were added forincubation at room temperature (RT) for 15 minutes. The luminescencesignal (for measurement of LDH release as indicative of cell death) wasread by EnVision (PerkinElmer). Data are presented as RelativeLuminescence Unit (RLU).

FIG. 5 shows the TCB-mediated specific T cell killing of RMF- orVLD-expressing target cells. We found that both 11D06-TCB and 33H09-TCBshowed specific killing on RMF peptide-pulsed T2 cells. 33F05-TCB didnot mediate specific killing of RMF or VLD peptide-pulsed cells. Inaddition, 13B04-TCB and 11B09-TCB mediated potent killing on VLDpeptide-pulsed T2 cells. 5C01-TCB showed killing of VLD-pulsed T2 cells,but also of RMF-pulsed cells at higher concentration, consistent withthe observations in the NFAT reporter assay (FIG. 4).

Example 7. T Cell Cytotoxicity Mediated by HLA-A2/WT1×CD3 BispecificAntibodies (“TCBs”) Upon Binding to WT1⁺ Cell Lines

To confirm the specific killing by the TCBs, we performed thecytotoxicity assay on WT1⁺ tumor cell lines. The HLA-A2+WT1+ cell lineswere SKM-1 cells (DZMZ No. ACC 547), and CHO cells transfected withHLA-A2/WT1_(RMF) complex (CHO-WT1) (in-house). The negative controlswere HLA-A2+WT1⁻ cells: BJAB (DZMZ No. ACC 757), ARH-77 (DZMZ No. ACC512), and CHO cells transfected with HLA-A2/MAGE-A4 complex (CHO-MAGEA4)(in-house), as well as a HLA-A2⁻WT1⁺ cell line: K562 (ATCC No. CLL-243)(FIG. 6A). The cytotoxicity assay was performed as described in Example6. Both 11D06-TCB and 33H09-TCB showed potent killing on SKM-1 andCHO-WT1 cells, but not on any HLA-A2⁺WT1⁻ cells and HLA-A2⁻WT1⁺ cells,indicating that these two TCBs have specificity for WT1 peptide RMF(FIG. 6 B, C). In contrast thereto, none of the VLD peptide-specificTCBs showed killing on HLA-A2⁺WT1⁺ cell lines, and if they showed lowpotency killing at high concentration (10 μg/ml) on the WT1⁺ cell lines,the same degree of killing was seen on WT1⁻ cell lines as well (FIG. 6D, E). Based on these functional data, we selected 11D06-TCB and33H09-TCB for further evaluation.

We also compared our selected TCBs with ESK1-TCB for their killingactivity of the tumor target cells. Interestingly, though ESK1-TCBachieved similar potency in mediating killing of SKM-1 cells as comparedto 11D06-TCB and 33H09-TCB, it also induced killing on HLA-A2⁺WT-1⁻ BJABcells, indicating that ESK1-TCB binding is not restricted toHLA-A2/WT1_(RMF) complex (FIG. 6F, G)

We tested the killing of multiple tumor cell lines that are HLA-A2+WT1+.Based on the EC₅₀ value for the killing, cell lines were categorizedinto those that were killed with an EC₅₀ of <1 μM (marked in Table 4with “++”), those that were killed with an EC₅₀ of >1 μM and <5 μM(marked in Table 4 with “+”), and those that were essentially not killed(marked in Table 4 with “no”).

All together, the selected TCBs 11D06-TCB and 33H09-TCB mediated T cellcytotoxicity on 6 out of 13 HLA-A2⁺WT1⁺ cell lines. The lack of killingon the other cell lines was likely due to the low expression of WT1presented on the cell surface by MHC I. Table 4 shows the results for11D06-TCB. The results for 33H09-TCB were similar (with EC₅₀ valuesbeing slightly higher than for 11D06-TCB).

TABLE 4 Killing of WT1+ cell lines by selected TCBs. EC50 (uM) Killingby in vitro 11D06-TCB killing by K-Ras Cell line name HLA-A2 WT1 Diseaseof Tissue Origin (RMF-specific) 11D06-TCB mutation SKM-1 + + Acutemyeloid leukemia ++ 0.09--0.35 yes T98G + + Glioblastoma ++ 0.8--1.7 noMDA-MB-231 + + Breast adenocarcinoma ++ 0.5--2.8 yes SW620 + +colorectal adenocarcinoma + 4.8 yes SW480 + + colorectaladenocarcinoma + 2.9 yes SET-2 + + Essential thrombocythemia (+)* iftreated  (0.005) no with Decitabine CTV-1 + + Leukemia, acute myeloid nono BV173 + + acute leukemia, chronic myeloid no no A-375 + + Melanoma nono LN-18 + + Glioblastoma no no U-266 + + myeloma no no OVCAR3 + +ovarian carcinoma no no Nalm6 + + B cell precursor leukemia no no

Example 8. T Cell Activation Mediated by HLA-A2/WT1×CD3 BispecificAntibodies (“TCBs”) Upon Binding to WT1⁺ Cell Lines

A prerequisite of TCB-mediated cytotoxicity on WT1⁺ target cells is thatT cells are activated to acquire effector function. We measured theactivation status of T cells by flow cytometry in the co-culture of Tcells and HLA-A2⁺WT1⁻ cells SKM-1 or HLA-A2⁺WT1⁻ cells BJAB in thepresence of the two selected TCBs, 33H09-TCB and 11D06-TCB, during thein vitro killing assay as described in Example 7. Cells were harvestedafter 18 hours of co-incubation, and stained with antibodies against CD3(Biolegend Cat. No. 300321), CD25 (Biolegend Cat. No. 302606) and CD69(Biolegend Cat. No. 310914) to measure T cell activation by flowcytometry.

As shown in FIG. 7, both TCBs induced up-regulation of CD69 and CD25 onCD3⁺ T cells upon binding to SKM-1 cells, but not BJAB cells, indicatingthat the specific recognition by TCBs of HLA-A2/WT1_(RMF) complexpresented by SKM-1 cells triggers CD3-mediated activation of T cells,eventually leading to the specific lysis of target cells.

Example 9. No Binding of Selected HLA-A2/WT1×CD3 Bispecific Antibodies(“TCBs”) to Reported Off-Target Peptides

A major potential risk in developing T cell receptor (TCR)-likeantibodies is the cross-reactivity of the antibody with peptidehomologues presented from proteins expressed by normal tissue. As it wasreported by Ataie et al. (J Mol Biol. (2016) 428:194-205) that ESK1antibody binds to sequence-similar peptides derived from protein MED13Land PIGQ, we tested our TCBs for their cross-reactivity to these twopeptides. The sequences of these peptides are shown in FIG. 8F, and SEQID NOs 79 and 80. In a flow cytometry-based binding assay, as describedin Example 4, we found that both 11D06-TCB and 33H09-TCB do not bind toPIGQ peptide. The binding to MED13L peptide by both TCBs is nearly 100fold less as compared to the native WT1 RMF peptide (FIG. 8 A, B).Although ESK1-TCB binds to RMF peptide in a low affinity, it binds toPIGQ peptide in a similar manner and even more strongly to MED13L (FIG.8C), consistent with the reported binding activity of the ESK1 IgG(Ataie et al., J Mol Biol. (2016) 428:194-205).

We also tested cross-reactivity using the NFAT-reporter assay asdescribed in Example 5. In line with the binding data, the strength ofNFAT activation on Jurkat cells upon binding to MED13L and PIGQ is atleast 100 fold weaker than the activation upon binding to WT1_(RMF)peptide by 11D06-TCB and 33H09-TCB (FIG. 8 D, E).

Example 10. No Binding of Selected HLA-A2/WT1×CD3 Bispecific Antibodies(“TCBs”) to Unidentified Off-Target Peptides

Cross-reactivity against homologous peptides (sharing key recognitionresidues) derived from normal tissue may cause serious and not readilypredictable toxicities by TCR-like antibody or TCR-based T cell therapy(see e.g. Linette et al, Blood (2013) 122:863-71; reporting fataltoxicity of MAGE A3-TCR-therapy due to off-target reactivity against aprotein expressed by cardiac tissue). Specificity of these agents istherefore critical. To thoroughly define specificity of our antibodies,based on the crystal structure data which confirms the positions in theRMF peptide involved in binding of 11D06 (see Example 15), we extendedour search for potential off-target peptides with similar amino acidsequence as WT1_(RMF) peptide (SEQ ID NO: 78) by masking RxxPNxxYx inthe peptidome databases (Swissprot and TrEMBL). We found additional 25peptides derived from different proteins which all have high predictedaffinity to HLA-A2 (Table 5). We then tested the cross-reactivity of ourselected TCBs on the peptide-pulsed T2 cells using the NFAT-reporterassay (peptide 1-6) or T cell cytotoxicity assay (peptide 7-25), asdescribed above.

For the first 6 peptides tested, there was no cross-reactivity to any ofthe 6 peptides by 11D06-TCB. 33H09-TCB showed some recognition toARHGEF11, but the degree of NFAT-activation was >100 fold lower ascompared to native RMF peptide (FIG. 9A, B). Similarly, thecross-reactivity to the other 19 off-target peptides was at least 100fold lower than to the native RMF peptide, as measured by direct T cellkilling of peptide-pulsed T2 cells (FIG. 9 C, D). This result wasconfirmed in a second experiment, wherein also ESK1-TCB was tested (FIG.9 E-G).

Taken together, we confirmed that 11D06 and 33H09 (but not the ESK1-likebinder) show specificity to the WT1_(RMF) peptide, and not to thepotential off-target peptides detected in the peptidome.

TABLE 5 Additional, newly identified off-target peptides. Peptide GeneSEQ Peptide sequence name ID NO  1 RLFPNLPEL ARHGEF11  81  2 RMFPNKYSLPRDM16  82  3 AMDPNAAYV SERPINA6  83  4 RMGPNIYEL NIPSNAP1  84  5NMPPNFPYI TAF3  85  6 YTIPNHPYL U4  86  7 RLFPNAKFL TPST1  87  8RMVPRAVYL IGFBP5  88  9 KMVPSIPYL RALGPS1  89 10 RIFPSYSYL ZNF382  90 11RLFPNSKFL TPST2  91 12 KMTPCIPYL RALGPS2  92 13 SMFPSLKYL TBCE  93 14RLLPSAPTL KIFC2  94 15 RLRPHVPYL SLC16A8  95 16 RMNPNSPSI ERH  96 17RVFNNRWYL ZBTB47  97 18 RLQLNNPYL MIOS  98 19 RMFFNGRYI ATP6V0A2  99 20RLSPNRPPL PKD1 100 21 ETFPNSWYL NAALADL1 101 22 GLKPNAIYL ROBO1 102 23RQFPNASLI RECQL 103 24 YIFPNCPFL SERPINA2 104 25 RLRINFPYL FAM220A 105

Example 11. No Cytotoxicity on CD34⁺ Stem Cells Mediated by SelectedHLA-A2/WT1×CD3 Bispecific Antibodies (“TCBs”)

It has been reported that CD34⁺ hematopoietic stem cells are WT1positive (Ramani and Cowell, J. Path (1996) 179:162-8), therefore it canpotentially be harmful if the TCBs would bind them. To study whetherCD34⁺ cells endogenously present the RMF peptide, we obtained HLA-A2⁺bone marrow derived CD34⁺ cells from 3 donors from Lonza, and tested ourselected TCBs in a killing assay as described above in Example 7.Whereas 11D06-TCB and 33H09-TCB mediate potent killing on SKM-1 cells,they had no killing activity on the CD34⁺ stem cells, indicating thatthese stem cells may not present the RMF peptide in the context ofHLA-A2 (FIG. 10).

Example 12. Definition of Binding Residues of Selected HLA-A2/WT1×CD3Bispecific Antibodies (“TCBs”) by Alanine Scan Assay

It is clear that our TCBs have different binding to RMF peptide andfunctional activity as compared to ESK1-TCB. To characterize thepotential binding motifs of the RMF peptide by the TCBs, we set up analanine scan assay using peptide arrays derived from native sequences byindividually replacing each amino acid with an alanine, and measured theNFAT-reporter signal by T2 cells pulsed with these peptides (FIG. 11A-L). Data are presented as RLU across increasing concentrations of TCB.We plotted the fold change of EC₅₀ relative to the EC₅₀ for the nativepeptide, and considered a fold change of ≥10 as significant, meaning therespective amino acid residue of the RMF peptide might be critical forrecognition by the 11D06-TCB and 33H09-TCB (FIG. 11M). We concluded thatboth 11D06-TCB and 33H09-TCB have critical contact with residues R1, F3,N5 and A6 (FIG. 11N), while ESK1 was shown to have close contact to RMFpeptide R1, P4 and N5 (Ataie et al., J Mol Biol. (2016) 428:194-205).

Example 13. Pharmacokinetic Profile of HLA-A2/WT1×CD3 BispecificAntibodies (“TCBs”) after Single Injection in NSG Mice

A single dose of 0.5 mg/kg of 33H09-TCB or 11D06-TCB was injected intoNSG mice. All mice were injected i.v. with 200 μl of the appropriatesolution. To obtain the proper amount of compounds per 200 μl, the stocksolution was diluted with histidine buffer. Three mice per time pointwere bled at 10 min, 1 hr, 3 hrs, 6 hrs, 24 hrs, 48 hrs, 72 hrs, 96 hrs,6 days, 8 days, 10 days and 12 days. The injected compound was analyzedin serum samples by ELISA. Biotinylated anti-huCD3-CDR antibody (RocheDiagnostics, Penzberg, Germany), test sample, digoxigenin-labelledanti-huFc antibody and anti-digoxigenin detection antibody (peroxidase(POD)) were added stepwise to a 96-well streptavidin-coated microtiterplate and incubated after every step for 1 h at room temperature. Theplate was washed three times after each step to remove unboundsubstances. Finally, the peroxidase-bound complex is visualized byadding ABTS (2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid)substrate solution to form a colored reaction product. The reactionproduct intensity, which was photometrically determined at 405 nm (withreference wavelength at 490 nm), is proportional to the analyteconcentration in the serum. The result (FIG. 12) showed a stablePK-behaviour for both clones tested which suggested a once weeklyschedule for subsequent efficacy studies.

Example 14. Efficacy Study with HLA-A2/WT1×CD3 Bispecific Antibodies(“TCBs”) in SKM-1 Xenograft in Humanized Mice

The first efficacy study of HLA-A2/WT1×CD3 bispecific antibodies wasaimed at comparing two different WT1 binders (11D06 and 33H09) in termsof efficacy in a human acute myeloid leukemia (AML) xenograft (SKM-1;HLA-A2+, WT-1+) in fully humanized NSG mice.

SKM-1 cells (human AML) were originally obtained from ATCC and depositedin the Roche Glycart internal cell bank. The cells were cultured inRPMI+10% FCS+1% glutamine in a water-saturated atmosphere at 5% CO₂. Invitro passage 15 was used for subcutaneous injection at a viability of98% with Matrigel (1:1 ratio).

Female NSG mice, age 4-5 weeks at start of the experiment (JacksonLaboratory) were maintained under specific-pathogen-free condition withdaily cycles of 12 h light/12 h darkness according to committedguidelines (GV-Solas; Felasa; TierschG). The experimental study protocolwas reviewed and approved by local government. After arrival, animalswere maintained for one week to get accustomed to the new environmentand for observation. Continuous health monitoring was carried out on aregular basis.

Female NSG mice were injected i.p. with 15 mg/kg of busulfan followedone day later by an i.v. injection of 1×10⁵ human hematopoietic stemcells isolated from cord blood. At week 14-16 after stem cell injectionmice were bled sublingual and blood was analyzed by flow cytometry forsuccessful humanization. Efficiently engrafted mice were randomizedaccording to their human T cell frequencies into the different treatmentgroups. At that time, mice were injected with SKM-1 tumor cells s.c. asillustrated in FIG. 13A and treated once weekly with the compounds orhistidine buffer (vehicle) when tumor size reached approximately 150 mm³(day 10). All mice were injected i.v. with 200 μl of the appropriatesolution. To obtain the proper amount of compounds per 200 μl, the stocksolutions were diluted with histidine buffer when necessary. Tumorgrowth was measured three times weekly using a caliper and tumor volumewas calculated as follows:

T_(v): (W²/2)×L (W: Width, L: Length)

Tumor growth Inhibition (TGI) as well as Tumor to control ratio (TCR)were calculated as follows:

${TGI}\text{:}\mspace{14mu} \frac{100 - {{Av}\left( {{T\_ treatment}^{\lbrack{{day}\mspace{14mu} x}\rbrack} - {T\_ treatment}^{\lbrack{baseline}\rbrack}} \right)}}{{Av}\left( {{T\_ Vehicle}^{\lbrack{{day}\mspace{14mu} x}\rbrack} - {T\_ Vehicle}^{\lbrack{baseline}\rbrack}} \right)}*100$${TCR}\text{:}\mspace{14mu} \frac{{Av}\left( {T\_ treatment}^{\lbrack{{day}\mspace{14mu} x}\rbrack} \right)}{{Av}\left( {T\_ Vehicle}^{\lbrack{{day}\mspace{14mu} x}\rbrack} \right)}$

FIG. 13 shows the tumor growth kinetics (mean) in all treatment groups(FIG. 13C) as well as the single tumor growth kinetics in each group(FIG. 13 D-F). As shown, both TCBs exhibit tumor growth inhibition, withclone 11D06 showing the strongest tumor growth inhibition with a TGI of101.3 (FIG. 13G).

Example 15. Crystal Structure of HLA-A2/WT1 Antibody/pMHC Complexes

Fab fragments were prepared by incubation of antibodies for 72 hours at25° C. in 50 mM Tris pH 8.0, 150 mM NaCl with 1.05 U plasmin (Roche,Cat. No. 602361) per mg. Cleaved Fc was separated from Fab fragmentsusing a 4.5 mL CaptureSelect CH1-affinity column (BAC BV, Cat. No.191.3120) equilibrated with 50 mM Tris, 100 mM glycine, 150 mM NaCl, pH8.0. Fab fragments were eluted from the column with 50 mM Tris, 100 mMglycine, 150 mM NaCl, pH 2.0 and neutralized with 0.5M sodium phosphatepH8.0 before loading on a size exclusion column S75 (GE Healthcare)equilibrated with 20 mM Tris, 150 mM NaCl, pH 7.4. Quality control wasperformed by doing analytical size exclusion (column Tosoh, TSK-GelG3000SWXL, on an Agilent HPLC 1200 system) and CE-SDS (Caliper LabChipGXII, Perkin Elmer) under non-reducing and reduced conditions. PurifiedFab fragments were frozen in liquid nitrogen and stored at −80° C.

Crystallization, Data Collection and Structure Determination of the 5C01Antibody/pMHC Complex

Crystallization.

The antibody/pMHC complex (Fab 5C01 HLA-A02/WT1_(VLD) pMHC) was preparedby mixing a 1.2-fold molar excess of HLA-A2/WT1 Fab fragment based onthe 5C01 binder (Fab 5C01) with HLA-A2/WT1_(VLD) peptide complex(HLA-A02/WT1_(VLD) pMHC). After 1 hour incubation at 4° C. the mixturewas concentrated to 10 mg/ml. Initial crystallization trials wereperformed in sitting drop vapor diffusion setups at 21° C. Crystalsappeared within 1 day out of 0.2 M sodium-tartrate, 20%polyethyleneglycol (PEG) 3350 with 10% 2-methyl-2,4-pentanediol (MPD)added to the crystallization droplet to improve crystal quality.Crystals were harvested directly from the screening plate without anyfurther optimization step.

Data Collection and Structure Determination.

For data collection crystals were flash frozen at 100K in precipitantsolution containing 15% glycerol. Diffraction data were collected at awavelength of 1.0000 Å using a PILATUS 6M detector at the beamline X10SAof the Swiss Light Source (Villigen, Switzerland). Data were processedwith XDS (Kabsch, W. Acta Cryst. D66, 133-144 (2010)) and scaled withSADABS (BRUKER). The crystals of the complex belong to space groupP2₁2₁2 with cell axes of a=158.94 Å, b=49.12 Å, c=128.63 Å and diffractto a resolution of 1.98 Å. The structure was determined by molecularreplacement with PHASER (McCoy, A. J, Grosse-Kunstleve, R. W., Adams, P.D., Storoni, L. C., and Read, R. J. J. Appl. Cryst. 40, 658-674 (2007))using the coordinates of the crystal structure with Protein Data Bank(PDB) entry 4N05 and an in house Fab structure as search models.Difference electron density was used to place peptide and to changeamino acids according to the sequence differences by real spacerefinement. Structures were refined with programs from the CCP4 suite(Collaborative Computational Project, Number 4 Acta Cryst. D50, 760-763(1994)) and BUSTER (Bricogne, G., Blanc, E., Brandi, M., Flensburg, C.,Keller, P., Paciorek, W., Roversi, P., Sharff, A., Smart, O. S.,Vonrhein, C., Womack, T. O. (2011). Buster version 2.9.5 Cambridge,United Kingdom: Global Phasing Ltd). Manual rebuilding was done withCOOT (Emsley, P., Lohkamp, B., Scott, W. G. and Cowtan, K. Acta CrystD66, 486-501 (2010)).

Data collection and refinement statistics are summarized in Table 6.

TABLE 6 Data collection and refinement statistics for Fab 5C01HLA-A02/WT1_(VLD) pMHC. 5C01-HLA-A02/ WT1_(VLD) pMHC Data collectionSpace group P2₁2₁2 Cell dimensions a, b, c (Å) 158.94, 49.12, 128.63 (°)90, 90, 90 Resolution (Å) 1.98 R_(sym) or R_(merge) 0.11 I/σI 12.01(0.53)  Completeness (%) 99.9 (99.9) Redundancy 6.61 (6.76) RefinementResolution (Å) 48.9-1.98 No. reflections 71103 R_(work)/R_(free)19.12/24.01 No. atoms Protein 6374 Water 528 B-factors Protein 66.32Water 59.27 R.m.s. deviations Bond lengths (Å) 0.010 Bond angles (°)1.07 *Values in parentheses are for highest-resolution shell.Structure of Fab 5C01 in Complex with HLA-A02/WT1_(VLD)

In order to characterize the interaction details of the VLD peptide withFab 5C01, the binding epitope and paratope of 5C01 with HLA-A02, wedetermined the crystal structure of the complex of 5C01 withHLA-A02/WT1_(VLD) pMHC at a resolution of 1.98 Å (FIG. 14A). Thestructure reveals Fab 5C01 to bind to pMHC by main contributions of theCDR1 and CDR3 of the light chain and by all CDRs of the heavy chain.From the VLD peptide (SEQ ID NO: 77), the side chains of residues Val1,Phe4 and Pro7 are in direct contact to the Fab. All other side chains ofthe peptide point towards the HLA-A02 molecule. Contribution of thepeptide to the contact surface area is ˜68 Å² whereas a total Fab-pMHCcontact area of ˜476 Å² is observed. A close-up of the Fab 5C01-pMHCinterface is shown in FIG. 15.

Analysis of the binding interface with the program PISA (E. Krissineland K. Henrick (2007), J. Mol. Biol. 372, 774-797) reveals aninteraction pattern of Fab 5C01 with the HLA-A02 via 8 hydrogen bonds,Pi-Pi interactions and van der Waals contacts. Side chain hydrogen bondsare formed between residues of heavy chain CDR3 (Trp97) and CDR2 (Ser52,Ser53) with Glu63 and Glu166 of HLA-A02. Further hydrogen bonds areestablished by light chain backbone atoms of Tyr91 and Ile93 withGln155. The complex is in addition stabilized via formation of Pi-Piinteractions of light chain residues Trp32 and Trp94 with HLA-A02 sidechains of Gln155 and His151. The N-terminal valine of the VLD peptideentertains hydrogen bonds through the backbone nitrogen to Tyr171 ofHLA-A02. Its side chain is oriented towards a pocket formed by Glu63 andTrp167 of HLA-A02 and Trp97 of the heavy chain of Fab 5C01. In additionPhe4 of the peptide makes edge to face interactions with Tyr100 of theheavy chain. A schematic Fab 5C01-pMHC interaction matrix summarizingthe contacts is shown in FIG. 16.

Crystallization, Data Collection and Structure Determination of the11D06 Antibody/pMHC Complex

Crystallization.

The antibody/pMHC complex (Fab 11D06 HLA-A02/WT1_(RMF) pMHC) wasprepared by mixing a 1:1 molar amount of HLA-A2/WT1 Fab fragment basedon the 11D06 binder (Fab 11D06) with HLA-A2/WT1_(RMF) peptide complex(HLA-A02/WT1_(RMF) pMHC). After 4 hours of incubation at 21° C. themixture was concentrated to 20 mg/ml. Initial crystallization trialswere performed in sitting drop vapor diffusion setups at 21° C. Crystalsappeared within 4 days out of 0.1 M Tris pH 8.0, 20% PEG 4000. Crystalswere harvested directly from the screening plate without any furtheroptimization step.

Data Collection and Structure Determination.

Data was collected, processed and scaled as described above. Thecrystals of the complex belong to space group P21 with cell axes ofa=54.11 Å, b=67.00 Å, c=139.36 Å with β=90.57° and diffract to aresolution of 2.64 Å. The structure was determined by molecularreplacement with PHASER using the coordinates of an in house Fab and MHCcomplex structure as search model. Difference electron density was usedto place peptide and to change amino acids according to the sequencedifferences by real space refinement. Structure refinement and manualrebuilding were done as described above.

Data collection and refinement statistics are summarized in Table 7.

TABLE 7 Data collection and refinement statistics for 11D06HLA-A02/WT1_(RMF) pMHC. 11D06 HLA-A02/ WT1_(RMF) pMHC Data collectionSpace group P2₁ Cell dimensions a, b, c (Å) 54.11, 67.00, 139.36 (°) 90,90.57, 90 Resolution (Å) 2.64 R_(sym) or R_(merge) 0.10 I/σI 13.10(0.82)  Completeness (%) 99.9 (99.8) Redundancy 3.79 (3.85) RefinementResolution (Å) 48.3-2.64 No. reflections 29606 R_(work)/R_(free)17.10/23.00 No. atoms Protein 6395 Water 250 B-factors Protein 67.52Water 57.46 R.m.s. deviations Bond lengths (Å) 0.010 Bond angles (°)1.20 *Values in parentheses are for highest-resolution shell.Structure of Fab 11D06 in Complex with HLA-A02/RMF pMHC

We determined the crystal structure of the complex of Fab 11D06 withHLA-A02/RMF pMHC at a resolution of 2.64 Å (FIG. 14B). The structureshows Fab 11D06 binds to pMHC by contributions of all CDRs. RMF peptide(SEQ ID NO: 78) side chains of residues Arg1, Met2, Pro4, Asn5, Ala6 andTyr8 are in direct contact to light and heavy chain of the Fab. Theremaining side chains of the peptide point towards the HLA-A02 molecule.Contribution of the peptide to the contact surface area is ˜107 Å². Thetotal Fab-pMHC contact area corresponds to ˜397 Å². A close-up of theFab 11D06-pMHC interface is shown in FIG. 17.

Analysis of the binding interface with the program PISA reveals aninteraction pattern of Fab 11D06 with HLA-A02 via 4 hydrogen bonds,numerous Pi-Pi interactions and van der Waals contacts. Hydrogen bondsare observed between residues of heavy chain CDR1 (Ser30, Ser31) andCDR3 (Gly100A) with Glu58 and Arg65 of HLA-A02. Further hydrogen bondsare established by light chain residue Asp50 with HLA-A02 Arg65. Besideothers, Trp100 of the heavy chain provides van der Waals and Pi-Picontacts to Arg65 and Lys66 of the HLA-A02 and to Pro4 of the RMFpeptide. The N-terminal arginine of the RMF peptide points with its sidechain into a polar pocket formed by Ser31, Glu97 and the backbonecarbonyl of Ile96 of the heavy chain of 11D06. Additional polar contactsto 11D06 and HLA-A02 are entertained by the RMF peptide residue Asn5which is part of a hydrogen bonding network to Trp32 of light chainCDR1, Glu92 of light chain CDR3 together with Gln155 of the HLA-A02. Aschematic Fab 11D06-pMHC interaction matrix summarizing the contacts isshown in FIG. 18.

3D Structure of ESK1 Retrieved from the Public Database:

For comparison, the publicly available crystal structure of antibody(Fab) ESK1 binding to HLA-A02/RMF pMHC (PDB ID 4WUU;http://www.rcsb.org/pdb/explore/explore.do?structureId=4WUU) wasanalyzed analogously with regard to its Fab-pMHC contacts. FIG. 14Cshows the Fab ESK1 with HLA-A02/RMF pMHC crystal structure from the sameangle (aligned on the HLA-A02 part) as the crystal structures of Fab5C01 with HLA-A02/VLD pMHC and Fab 11D06 with HLA-A02/RMF pMHC (FIGS. 14A and B). A close-up of the Fab ESK1-pMHC interface is shown in FIG. 19,and a schematic Fab ESK1-pMHC interaction matrix summarizing thecontacts is shown in FIG. 20.

The structural comparison reveals that 5C01 and particularly 11D06 coverand bind to a larger fraction of the respective WT1 peptide than ESK1,which forms specific contacts exclusively with the N-terminal Arg of theRMF peptide while the remainder of the binding interface is provided byHLA-A02. Based on these observations one can conclude that 5C01 and11D06 should be less likely to tolerate off-target peptides than ESK1 asthey create significantly more steric hindrance for peptides withnon-VLD or non-RMF-like sidechains on the exposed positions.

All graphical representations of the crystal structures were createdwith BIOVIA Discovery Studio 4.5, Dassault Systemes BIOVIA.

Epitope and Paratope Residues (5 Å Radius)

A summary of the epitope and paratope for the binders 5C01, 11D06 andESK1 is shown below. Epitope and paratope (definition based on 5 Åneighborhood radius) residues are highlighted in bold italic script. CDRresidues are highlighted with grey background.

HLA-A02 1-60 5C01 11D06 ESK1

61-120 5C01 11D06 ESK1

121-180 5C01 11D06 ESK1

181-240 5C01RTDAPKTHMTHHAVSDHEATLRCWALSFYPAEITLTWQRDGEDQTQDTELVETRPAGDGT 11D06RTDAPKTHMTHHAVSDHEATLRCWALSFYPAEITLTWQRDGEDQTQDTELVETRPAGDGT ESK1RTDAPKTHMTHHAVSDHEATLRCWALSFYPAEITLTWQRDGEDQTQDTELVETRPAGDGT 241-2755C01 FQKWAAVVVPSGQEQRYTCHVQHEGLPKPLTLRWE 11D06FQKWAAVVVPSGQEQRYTCHVQHEGLPKPLTLRWE ESK1FQKWAAVVVPSGQEQRYTCHVQHEGLPKPLTLRWE Peptide 1-9 5C01 11D06 ESK1

Fab Heavy Chain  1-60 5C01 11D06 ESK1

61-120 5C01 11D06 ESK1

121-180 5C01SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS 11D06SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS ESK1SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS 181-2245C01 SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC 11D06SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC ESK1SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKS Fab Light Chain  1-60 5C0111D06 ESK1

61-120 5C01 11D06 ESK1

121-180 5C01VFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYS 11D06VFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYS ESK1TVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADGSPVKAGVETTKPSKQSNNKYA 181-2205C01 LSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 11D06LSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC ESK1ASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS

Residues Involved in Chemical Interactions

A summary of the residues involved in chemical interaction for thebinders 5C01, 11D06 and

ESK1 is shown below. Residues involved in specific chemical interactions(compare FIGS. 16, 18 and 20) are highlighted in bold italic script. CDRresidues are highlighted with grey background.

HLA-A02 1-60 5C01 11D06 ESK1

61-120 5C01 11D06 ESK1

121-180 5C01 11D06 ESK1

181-240 5C01RTDAPKTHMTHHAVSDHEATLRCWALSFYPAEITLTWQRDGEDQTQDTELVETRPAGDGT 11D06RTDAPKTHMTHHAVSDHEATLRCWALSFYPAEITLTWQRDGEDQTQDTELVETRPAGDGT ESK1RTDAPKTHMTHHAVSDHEATLRCWALSFYPAEITLTWQRDGEDQTQDTELVETRPAGDGT 241-2755C01 FQKWAAVVVPSGQEQRYTCHVQHEGLPKPLTLRWE 11D06FQKWAAVVVPSGQEQRYTCHVQHEGLPKPLTLRWE ESK1FQKWAAVVVPSGQEQRYTCHVQHEGLPKPLTLRWE Peptide 1-9 5C01 11D06 ESK1

Fab Heavy Chain  1-60 5C01 11D06 ESK1

61-120 5C01 11D06 ESK1

121-180 5C01SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS 11D06SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS ESK1SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS 181-2245C01 SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC 11D06SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC ESK1SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKS Fab Light Chain  1-60 5C0111D06 ESK1

61-120 5C01 11D06 ESK1

121-180 5C01VFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYS 11D06VFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYS ESK1TVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADGSPVKAGVETTKPSKQSNNKYA 181-2205C01 LSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 11D06LSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC ESK1ASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS

Example 16. Comparison of HLA-A2/WT1×CD3 Bispecific Antibodies (“TCBs”)with Different CD3 Binders

We compared the potency of killing activities of 11D06-TCB withdifferent CD3 binders, using SKM-1 cells in an assay as described above(Example 7). CH2527 is the CD3 binder of the TCBs used in the previousexperiments (see SEQ ID NOs 121 and 122 for the VH and VL sequences). V9is a CD3 binder described Rodrigues et al., Int J Cancer Suppl (1992) 7,45-50, and WO 1992/22653 (see SEQ ID NOs 136 and 137 for the VH and VLsequences), and 40G5C is described in WO 2015/095392 (see SEQ ID NOs 184and 185 (“hu40G5c”) of WO 2015/095392 for the VH and VL sequences).

The affinity of CD3 binders CH2527, 40G5C and V9 are shown in Table 8.

As shown in FIG. 21, we observed that 11D06-TCB (CH2527) showed the samepotency of T cell-mediated killing on HLA-A2⁺WT1⁺ SKM-1 cell lines as11D06-TCB (V9), whereas 11D06-TCB (40G5C) showed strongly reducedkilling potency.

TABLE 8 Affinities of CD3 binders compared in this experiment. Affinity(Kd) CD3 clone Cross-reactivity [nM] CH2527 Hu-Cyno  85-130 40G5CHu-Cyno 390-460 V9 Hu only 35-50

Example 17. T Cell Cytotoxicity Mediated by HLA-A2/WT1×CD3 BispecificAntibodies (“TCBs”) Upon Binding to RMF Peptide-Pulsed T2 Cells

The killing activity of 11D06-TCB (V9) (see SEQ ID NOs 123, 125, 139,140 for full sequences) was compared to analogous TCBs with differentHLA/WT1 binders (“Aali” and “Daniel”, see WO 2017/060201, SEQ ID NOs 5(VH) and 6 (VL) and SEQ ID NOs 35 (VH) and 36 (VL), respectively). TheESK1-TCB and the untargeted DP47-TCB were also included as controls. Theexperiment was performed as described in Example 6. The RMFpeptide-pulsed cells (100 2×10⁵ cells/ml) were co-cultured with 50 μl ofT cells (2×10⁶ cells/ml), and with serial dilutions of TCB (50 μl) for24 hours at 37° C. with 5% CO₂.

FIG. 22 shows the TCB-mediated specific T cell killing of RMF-expressingtarget cells. We found that 11D06-TCB and ESK1-TCB mediated potentkilling on RMF peptide-pulsed T2 cells, whereas the TCBs based on theHLA/WT1 binders “Aali” and “Daniel” (Aali-TCB and Daniel-TCB) showedweak killing of RMF-pulsed cells only at the highest concentrations.

Example 18. No Binding of Selected HLA-A2/WT1×CD3 Bispecific Antibodies(“TCBs”) to Off-Target Peptides

We also compared the cross-reactivity of 11D06-TCB (V9), Aali-TCB,Daniel-TCB and ESK1-TCB with the off-target peptides described inExamples 9 and 10.

For this experiment, Jurkat NFAT reporter cells expressing ananti-PGLALA chimeric antigen receptor (CAR) were used (CAR J assay, seePCT application claiming priority from European patent application no.EP17209201.7, incorporated herein by reference in its entirety). Theanti-PGLALA CAR recognizes the P329G L234A L235A (“PGLALA”, EUnumbering) mutations in the Fc region of the TCBs. Peptide-pulsed T2cells as described above were used as target cells. The principle of theassay is to co-culture the Jurkat-NFAT engineered effector cells withtarget cells. Only upon simultaneous binding of the TCBs to the CAR (viathe PGLALA mutation) and the target antigen, the NFAT promoter isactivated and leads to increasing luciferase expression in the Jurkateffector cells. Upon addition of an adequate substrate, active FireflyLuciferase leads to emission of luminescence, which can be measured as asignal of CAR-mediated activation.

Briefly, target cells were harvested and viability determined. 20 000target cells/well were plated in a round-bottom, 96-well-plate (Greinerbio-one, #650180) in 100 μl medium. Non-pulsed T2 cells were used asnegative control. 50 μl/well of diluted TCB were added to the targetcells. Subsequently, Jurkat-NFAT reporter cells were harvested andviability assessed, resuspended in cell culture medium and added totumor cells at 100 000 cells/well (50 μl/well) to obtain a finaleffector-to-target (E:T) ratio of 5:1 and a final volume of 200 μl perwell. Cells were incubated for 20 h at 37° C. in a humidified incubator.At the end of the incubation time, the plates were adapted to roomtemperature (about 15 min). 125 ul of media/well was removed from thetop and 25 μl/well of One-Glo Luciferase (Promega #E6120) was added, andmixed. 100 ul/well of the mixture was then transferred to 96 well flatbottom plate (Greiner bio-one, #655098) and the plate was incubated for15 min in the dark before luminescence was detected using Perkin Elmer.

As shown in FIG. 23, Aali-TCB (A), Daniel-TCB (B) and ESK1-TCB (C)resulted in weaker activation of Jurkat NFAT reporter cell line(corresponding to weaker killing shown in FIG. 22) and moreover, asmaller window between recognition of RMF peptide and other off-targetpeptides comparing to 11D06-TCB (V9) (D).

Example 19. Pharmacokinetic Profile of HLA-A2/WT1×CD3 BispecificAntibody (11D06-TCB (V9)) after Single Injection in NSG Mice

A single dose of 1 mg/kg of 11D06-TCB (V9) was injected into humanizedand tumor-bearing NSG mice. Mice were injected i.v. with 200 μl of TCB,diluted with histidine buffer. Three mice per time point were bled at 10min, 6 h, 24 h, 72 h and 7 days. The injected compound was analyzed inserum samples by ELISA as described in Example 13.

The result (FIG. 24) showed a stable PK-behaviour for the tested TCB,which suggested a once weekly schedule for subsequent efficacy studies.

Example 20. Efficacy Study with HLA-A2/WT1×CD3 Bispecific Antibody(11D06-TCB (V9)) in SKM-1 Xenograft in Humanized Mice

This efficacy study was aimed to evaluate the efficacy of 11D06-TCB (V9)in a human AML xenograft (SKM-1) in fully humanized NSG mice. Theexperiment was performed as described in Example 14.

FIG. 25 shows the tumor growth kinetics (mean, FIG. 25A) as well as thesingle tumor growth kinetics in each group (FIG. 25 B, C). As shown, theTCB exhibits tumor growth inhibition a TGI of 78 at study day 48 (FIG.25D).

Example 21. Comparison of HLA-A2/WT1×CD3 Bispecific Antibodies (“TCBs”)with Different Molecular Formats

We compared the activity of 11D06-TCB (V9) with an analogous molecule ina “1+1 CrossMab” format (as depicted in FIG. 1A). Activity was monitoredusing a cell-based functional assay which detects TCB-mediatedactivation of a reporter cell line (Jurkat NFAT) in the presence oftarget cells in a dose-dependent manner. The assay was performedessentially as described in Example 5 above, using as target cellsCHO-K1 cells expressing a HLA-A02/WT1_(RMF) pMHC complex. Reporter cellactivation occurs upon simultaneous binding of the TCB to theHLA-A02/WT1_(RMF) pMHC complex on target cells and to the CD3c unit of Tcell receptor (TCR) on reporter cells, which leads to hyper-clusteringof CD3 and thereby to TCR activation. Subsequent initiation ofcorresponding intracellular signaling pathways results in activation oftranscription factor NFAT which induces expression of a NFAT-drivenluciferase reporter gene. Activity of luciferase reporter is measuredupon addition of substrate by luminescence read-out.

The result of this experiment is shown in FIG. 26. Relative light units(RLU) reflecting target-dependent reporter cell activation are plottedagainst antibody concentration. As can be seen in FIG. 26, the moleculein the “1+1” format showed a lower activity than the “2+1” format inthis assay.

Amino Acid Sequences

SEQ Sequence ID NO 11D06 SYAIS   1 HCDR1 11D06 GIIPIFGTANYAQKFQG   2HCDR2 11D06 SIELWWGGFDY   3 HCDR3 11D06 RASQSISSWLA   4 LCDR1 11D06DASSLES   5 LCDR2 11D06 QQYEDYTT   6 LCDR3 11D06 VHQVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQA   7PGQGLEWMGGIIPIFGTANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCARSIELWWGGFDYWGQGTTVTVSS 11D06 VLDIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKP   8GKAPKLLIYDASSLESGVPSRFSGSGSGTEFTLTIGSLQP DDFATYYCQQYEDYTTFGQGTKVEIK33H09 SYAIS   9 HCDR1 33H09 GIIPIFGTANYAQKFQG  10 HCDR2 33H09 GSYDLFSLDY 11 HCDR3 33H09 RASQSISSWLA  12 LCDR1 33H09 DASSLES  13 LCDR2 33H09QQYYDGIT  14 LCDR3 33H09 VH QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQA  15PGQGLEWMGGIIPIFGTANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCARGSYDLFSLDYWGQGTTVTVSS 33H09 VLDIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKP  16GKAPKLLIYDASSLESGVPSRFSGSGSGTEFTLTISSLQP DDFATYYCQQYYDGITFGQGTKVEIK13B04 SYYWS  17 HCDR1 13B04 YIYYSGSTNYNPSLKS  18 HCDR2 13B04 VSYNGLDY 19 HCDR3 13B04 RASQSISSWLA  20 LCDR1 13B04 DASSLES  21 LCDR2 13B04QQYNMWNPVT  22 LCDR3 13B04 VH EVQLLESGPGLVKPSETLSLTCTVSGGSISSYYWSWIRQP 23 PGKGLEWIGYIYYSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARVSYNGLDYWGQGTLVTVSS 13B04 VLDIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKP  24GKAPKLLIYDASSLESGVPSRFSGSGSGTEFTLTISSLQP DDFATYYCQQYNMWNPVTFGQGTKVEIK11B09 SYAIS  25 HCDR1 11B09 GIIPIFGTANYAQKFQG  26 HCDR2 11B09VPGRWYGAMDY  27 HCDR3 11B09 RASQSISSWLA  28 LCDR1 11B09 DASSLES  29LCDR2 11B09 QQEDDYPLT  30 LCDR3 11B09 VHQVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQA  31PGQGLEWMGGIIPIFGTANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCARVPGRWYGAMDYWGQGTTVTVSS 11B09 VLDIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKP  32GKAPKLLIYDASSLESGVPSRFSGSGSGTEFTLTISSLQP DDFATYYCQQEDDYPLTFGQGTKVEIK33F05 SYYWS  33 HCDR1 33F05 YIYYSGSTNYNPSLKS  34 HCDR2 33F05 SYYEAFDY 35 HCDR3 33F05 LCDR1 QGDSLRSYYAS  36 33F05 LCDR2 GKNNRPS  3733F05 LCDR3 NSPDMNGNAV  38 33F05 VHQVQLQESGPGLVKPSETLSLTCTVSGGSINSYYWSWIRQP  39PGKGLEWIGYIYYSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARSYYEAFDYWGQGTLVTVSS 33F05 VLSSELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPG  40QAPVLVIYGKNNRPSGIPDRFSGSSSGNTASLTITGAQAE DEADYYCNSPDMNGNAVFGGGTKLTVL5E11 HCDR1 SYAIS  41 5E11 HCDR2 GIIPIFGTANYAQKFQG  42 5E11 HCDR3SSYDLYSFDY  43 5E11 LCDR1 RASQSISSWLA  44 5E11 LCDR2 DASSLES  455E11 LCDR3 QQYSFPPMIT  46 5E11 VHQVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQA  47PGQGLEWMGGIIPIFGTANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCARSSYDLYSFDYWGQGTTVTVSS 5E11 VLDIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKP  48GKAPKLLIYDASSLESGVPSRFSGSGSGTEFTLTISSLQP DDFATYYCQQYSFPPMITFGQGTKVEIK13E08 SYAMS  49 HCDR1 13E08 AISGSGGSTYYADSVKG  50 HCDR2 13E08 TYPYTGSFDY 51 HCDR3 13E08 LCDR1 RASQSISSWLA  52 13E08 LCDR2 DASSLES  5313E08 LCDR3 QQNYNYPPT  54 13E08 VHEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQA  55PGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKTYPYTGSFDYWGQGTLVTVSS 13E08 VLDIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKP  56GKAPKLLIYDASSLESGVPSRFSGSGSGTEFTLTISSLQP DDFATYYCQQNYNYPPTFGQGTKVEIK5C01 HCDR1 SYAMS  57 5C01 HCDR2 AISGSGGSTYYADSVKG  58 5C01 HCDR3GSWVSYAFDY  59 5C01 LCDR1 RASQSISSWLA  60 5C01 LCDR2 DASSLES  615C01 LCDR3 QQYSIWFPYT  62 5C01 VHEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQA  63PGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGSWVSYAFDYWGQGTLVTVSS 5C01 VLDIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKP  64GKAPKLLIYDASSLESGVPSRFSGSGSGTEFTLTISSLQP DDFATYYCQQYSIWFPYTFGQGTKVEIK11G06 SYAIS  65 HCDR1 11G06 GIIPIFGTANYAQKFQG  66 HCDR2 11G06 TGPYYGAFDY 67 HCDR3 11G06 RASQSISSWLA  68 LCDR1 11G06 DASSLES  69 LCDR2 11G06QQGFRGYT  70 LCDR3 11G06 VH QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQA  71PGQGLEWMGGIIPIFGTANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCARTGPYYGAFDYWGQGTTVTVSS 11G06 VLDIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKP  72GKAPKLLIYDASSLESGVPSRFSGSGSGTEFTLTISSLQP DDFATYYCQQGFRGYTFGQGTKVEIKESK1 VH QMQLVQSGAEVKEPGESLRISCKGSGYSFTNFWISWVRQM  73PGKGLEWMGRVDPGYSYSTYSPSFQGHVTISADKSTSTAYLQWNSLKASDTAMYYCARVQYSGYYDWFDPWGQGTLVTVS S ESK1 VLQAWTQPPSASGTPGQRVTISCSGSSSNIGSNTVNWYQQVP  74GTAPKLLIYSNNQRPSGVPDRFSGSKSGTSASLAISGLQS EDEADYYCAAWDDSLNGWVFGGGTKLTVLUntargeted EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQA  75 VHPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGSGFDYWGQGTLVTVSS Untargeted VLEIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQK  76PGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLE PEDFAVYYCQQYGSSPLTFGQGTKVEIKVLD peptide VLDFAPPGA  77 RMF peptide RMFPNAPYL  78 MED13L RMFPTPPSL  79PIGQ RMFPGEVAL  80 ARHGEF11 RLFPNLPEL  81 PRDM16 RMFPNKYSL  82 SERPINA6AMDPNAAYV  83 NIPSNAP1 RMGPNIYEL  84 TAF3 NMPPNFPYI  85 U4 YTIPNHPYL  86TPST1 RLFPNAKFL  87 IGFBP5 RMVPRAVYL  88 RALGPS1 KMVPSIPYL  89 ZNF382RIFPSYSYL  90 TPST2 RLFPNSKFL  91 RALGPS2 KMTPCIPYL  92 TBCE SMFPSLKYL 93 KIFC2 RLLPSAPTL  94 SLC16A8 RLRPHVPYL  95 ERH RMNPNSPSI  96 ZBTB47RVFNNRWYL  97 MIOS RLQLNNPYL  98 ATP6V0A2 RMFFNGRYI  99 PKD1 RLSPNRPPL100 NAALADL1 ETFPNSWYL 101 ROBO1 GLKPNAIYL 102 RECQL RQFPNASLI 103SERPINA2 YIFPNCPFL 104 FAM220A RLRINFPYL 105 Human WT1MGSDVRDLNALLPAVPSLGGGGGCALPVSGAAQWAPVLDF 106APPGASAYGSLGGPAPPPAPPPPPPPPPHSFIKQEPSWGGAEPHEEQCLSAFTVHFSGQFTGTAGACRYGPFGPPPPSQASSGQARMFPNAPYLPSCLESQPAIRNQGYSTVTFDGTPSYGHTPSHHAAQFPNHSFKHEDPMGQQGSLGEQQYSVPPPVYGCHTPTDSCTGSQALLLRTPYSSDNLYQMTSQLECMTWNQMNLGATLKGVAAGSSSSVKWTEGQSNHSTGYESDNHTTPILCGAQYRIHTHGVFRGIQDVRRVPGVAPTLVRSASETSEKRPFMCAYPGCNKRYFKLSHLQMHSRKHTGEKPYQCDFKDCERRFSRSDQLKRHQRRHTGVKPFQCKTCQRKFSRSDHLKTHTRTHTGKTSEKPFSCRWPSCQKKFARSDELVRHHNMHQR NMTKLQLAL Human CD3MQSGTHWRVLGLCLLSVGVWGQDGNEEMGGITQTPYKVSI 107SGTTVILTCPQYPGSEILWQHNDKNIGGDEDDKNIGSDEDHLSLKEFSELEQSGYYVCYPRGSKPEDANFYLYLRARVCENCMEMDVMSVATIVIVDICITGGLLLLVYYWSKNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPNPDYEPIRKGQRDLYS GLNQRRI CynomolgusMQSGTRWRVLGLCLLSIGVWGQDGNEEMGSITQTPYQVSI 108 CD3SGTTVILTCSQHLGSEAQWQHNGKNKEDSGDRLFLPEFSEMEQSGYYVCYPRGSNPEDASHHLYLKARVCENCMEMDVMAVATIVIVDICITLGLLLLVYYWSKNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPNPDYEPIRKGQQDLYSGLNQRRI hIgG1 FcDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVT 109 regionCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQG NVFSCSVMHEALHNHYTQKSLSLSPlinker GGGGSGGGGS 110 linker DGGGGSGGGGS 111 Human kappaRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQ 112 CL domainWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYE KHKVYACEVTHQGLSSPVTKSFNRGECHuman QPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVA 113 lambda CLWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKS domainHRSYSCQVTHEGSTVEKTVAPTECS Human IgG1ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS 114 heavy chainWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT constantYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGG region (CH1-PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNW CH2-CH3)YVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSP CD3 HCDR1 TYAMN 115CD3 HCDR2 RIRSKYNNYATYYADSVKG 116 CD3 HCDR3 HGNFGNSYVSWFAY 117 CD3 LCDR1GSSTGAVTTSNYAN 118 CD3 LCDR2 GTNKRAP 119 CD3 LCDR3 ALWYSNLWV 120 CD3 VHEVQLLESGGGLVQPGGSLRLSCAASGFTFSTYAMNWVRQA 121PGKGLEWVSRIRSKYNNYATYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCVRHGNFGNSYVSWFAYWGQGTL VTVSS CD3 VLQAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNYANWVQE 122KPGQAFRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGA QPEDEAEYYCALWYSNLWVFGGGTKLTVLWT1 11D06 QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQA 123 VH-CH1(EE)-PGQGLEWMGGIIPIFGTANYAQKFQGRVTITADKSTSTAY Fc(hole,MELSSLRSEDTAVYYCARSIELWWGGFDYWGQGTTVTVSS PGLALA)ASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSP WT1 11D06QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQA 124 VH-CH1(EE)-PGQGLEWMGGIIPIFGTANYAQKFQGRVTITADKSTSTAY CD3 VL-CH1-MELSSLRSEDTAVYYCARSIELWWGGFDYWGQGTTVTVSS Fc(knob,ASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVS PGLALA)WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDGGGGSGGGGSQAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNYANWVQEKPGQAFRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGAQPEDEAEYYCALWYSNLWVFGGGTKLTVLSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP WT1 11D06DIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKP 125 VL-CL(RK)GKAPKLLIYDASSLESGVPSRFSGSGSGTEFTLTIGSLQPDDFATYYCQQYEDYTTFGQGTKVEIKRTVAAPSVFIFPPSDRKLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGL SSPVTKSFNRGEC WT1 33H09QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQA 126 VH-CH1(EE)-PGQGLEWMGGIIPIFGTANYAQKFQGRVTITADKSTSTAY Fc(hole,MELSSLRSEDTAVYYCARGSYDLFSLDYWGQGTTVTVSSA PGLALA)STKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ KSLSLSP WT1 33H09QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQA 127 VH-CH1(EE)-PGQGLEWMGGIIPIFGTANYAQKFQGRVTITADKSTSTAY CD3 VL-CH1-MELSSLRSEDTAVYYCARGSYDLFSLDYWGQGTTVTVSSA Fc(knob,STKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSW PGLALA)NSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDGGGGSGGGGSQAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNYANWVQEKPGQAFRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGAQPEDEAEYYCALWYSNLWVFGGGTKLTVLSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP WT1 33H09DIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKP 128 VL-CL(RK)GKAPKLLIYDASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYYDGITFGQGTKVEIKRTVAAPSVFIFPPSDRKLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGL SSPVTKSFNRGEC CD3 VH-CLEVQLLESGGGLVQPGGSLRLSCAASGFTFSTYAMNWVRQA 129PGKGLEWVSRIRSKYNNYATYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSSASVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC CD3 HCDR1 GYTMN 130 (V9) CD3 HCDR2LINPYKGVSTYNQKFKD 131 (V9) CD3 HCDR3 SGYYGDSDWYFDV 132 (V9) CD3 LCDR1RASQDIRNYLN 133 (V9) CD3 LCDR2 YTSRLES 134 (V9) CD3 LCDR3 QQGNTLPWT 135(V9) CD3 VH (V9) EVQLVESGGGLVQPGGSLRLSCAASGYSFTGYTMNWVRQA 136PGKGLEWVALINPYKGVSTYNQKFKDRFTISVDKSKNTAYLQMNSLRAEDTAVYYCARSGYYGDSDWYFDVWGQGTLVTV SS CD3 VL (V9)DIQMTQSPSSLSASVGDRVTITCRASQDIRNYLNWYQQKP 137GKAPKLLIYYTSRLESGVPSRFSGSGSGTDYTLTISSLQP EDFATYYCQQGNTLPWTFGQGTKVEIKHLA-A2 GSHSMRYFFTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDA 138ASQRMEPRAPWIEQEGPEYWDGETRKVKAHSQTHRVDLGTLRGYYNQSEAGSHTVQRMYGCDVGSDWRFLRGYHQYAYDGKDYIALKEDLRSWTAADMAAQTTKHKWEAAHVAEQLRAYLEGTCVEWLRRYLENGKETLQRTDAPKTHMTHHAVSDHEATLRCWALSFYPAEITLTWQRDGEDQTQDTELVETRPAGDGTFQKWAAVVVPSGQEQRYTCHVQHEGLPKPLTLRWE WT1 11D06QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQA 139 VH-CH1(EE)-PGQGLEWMGGIIPIFGTANYAQKFQGRVTITADKSTSTAY CD3 (V9) VL-MELSSLRSEDTAVYYCARSIELWWGGFDYWGQGTTVTVSS CH1-Fc(knob,ASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVS PGLALA)WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDIRNYLNWYQQKPGKAPKLLIYYTSRLESGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGNTLPWTFGQGTKVEIKSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDK SRWQQGNVFSCSVMHEALHNHYTQKSLSLSPCD3 (V9) EVQLVESGGGLVQPGGSLRLSCAASGYSFTGYTMNWVRQA 140 VH-CLPGKGLEWVALINPYKGVSTYNQKFKDRFTISVDKSKNTAYLQMNSLRAEDTAVYYCARSGYYGDSDWYFDVWGQGTLVTVSSASVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKAD YEKHKVYACEVTHQGLSSPVTKSFNRGEC

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, the descriptions and examples should not be construed aslimiting the scope of the invention. The disclosures of all patent andscientific literature cited herein are expressly incorporated in theirentirety by reference.

1. An antibody that binds to HLA-A2/WT1, wherein the antibody binds anepitope of HLA-A2/WT1, particularly HLA-A2/WT1_(RMF), comprising atleast three amino acid residues of the WT1 peptide in HLA-A2/WT1,particularly the WT1_(RMF) peptide.
 2. The antibody of claim 1, whereinsaid at least three amino acid residues are selected from the amino acidresidues corresponding to amino acid residues R1, M2, P4, N5, A6 and Y8of the WT1_(RMF) peptide.
 3. An antibody that binds to HLA-A2/WT1,wherein the antibody comprises (i) a heavy chain variable region (VH)comprising a heavy chain complementary determining region (HCDR) 1 ofSEQ ID NO: 1, a HCDR 2 of SEQ ID NO: 2, and a HCDR 3 of SEQ ID NO: 3,and a light chain variable region (VL) comprising a light chaincomplementarity determining region (LCDR) 1 of SEQ ID NO: 4, a LCDR 2 ofSEQ ID NO: 5 and a LCDR 3 of SEQ ID NO: 6; (ii) a VH comprising a HCDR 1of SEQ ID NO: 9, a HCDR 2 of SEQ ID NO: 10, and a HCDR 3 of SEQ ID NO:11, and a VL comprising a LCDR 1 of SEQ ID NO: 12, a LCDR 2 of SEQ IDNO: 13 and a LCDR 3 of SEQ ID NO: 14; (iii) a VH comprising a HCDR 1 ofSEQ ID NO: 17, a HCDR 2 of SEQ ID NO: 18, and a HCDR 3 of SEQ ID NO: 19,and a VL comprising a LCDR 1 of SEQ ID NO: 20, a LCDR 2 of SEQ ID NO: 21and a LCDR 3 of SEQ ID NO: 22; (iv) a VH comprising a HCDR 1 of SEQ IDNO: 25, a HCDR 2 of SEQ ID NO: 26, and a HCDR 3 of SEQ ID NO: 27, and aVL comprising a LCDR 1 of SEQ ID NO: 28, a LCDR 2 of SEQ ID NO: 29 and aLCDR 3 of SEQ ID NO: 30; (v) a VH comprising a HCDR 1 of SEQ ID NO: 33,a HCDR 2 of SEQ ID NO: 34, and a HCDR 3 of SEQ ID NO: 35, and a VLcomprising a LCDR 1 of SEQ ID NO: 36, a LCDR 2 of SEQ ID NO: 37 and aLCDR 3 of SEQ ID NO: 38; (vi) a VH comprising a HCDR 1 of SEQ ID NO: 41,a HCDR 2 of SEQ ID NO: 42, and a HCDR 3 of SEQ ID NO: 43, and a VLcomprising a LCDR 1 of SEQ ID NO: 44, a LCDR 2 of SEQ ID NO: 45 and aLCDR 3 of SEQ ID NO: 46; (vii) a VH comprising a HCDR 1 of SEQ ID NO:49, a HCDR 2 of SEQ ID NO: 50, and a HCDR 3 of SEQ ID NO: 51, and a VLcomprising a LCDR 1 of SEQ ID NO: 52, a LCDR 2 of SEQ ID NO: 53 and aLCDR 3 of SEQ ID NO: 54; (viii) a VH comprising a HCDR 1 of SEQ ID NO:57, a HCDR 2 of SEQ ID NO: 58, and a HCDR 3 of SEQ ID NO: 59, and a VLcomprising a LCDR 1 of SEQ ID NO: 60, a LCDR 2 of SEQ ID NO: 61 and aLCDR 3 of SEQ ID NO: 62; or (ix) a VH comprising a HCDR 1 of SEQ ID NO:65, a HCDR 2 of SEQ ID NO: 66, and a HCDR 3 of SEQ ID NO: 67, and a VLcomprising a LCDR 1 of SEQ ID NO: 68, a LCDR 2 of SEQ ID NO: 69 and aLCDR 3 of SEQ ID NO:
 70. 4. The antibody of claim 3, wherein theantibody comprises (i) a VH comprising an amino acid sequence that is atleast about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acidsequence of SEQ ID NO: 7, and a VL comprising an amino acid sequencethat is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to theamino acid sequence of SEQ ID NO: 8; (ii) a VH comprising an amino acidsequence that is at least about 95%, 96%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO: 15, and a VLcomprising an amino acid sequence that is at least about 95%, 96%, 97%,98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 16;(iii) a VH comprising an amino acid sequence that is at least about 95%,96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQID NO: 23, and a VL comprising an amino acid sequence that is at leastabout 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acidsequence of SEQ ID NO: 24; (iv) a VH comprising an amino acid sequencethat is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to theamino acid sequence of SEQ ID NO: 31, and a VL comprising an amino acidsequence that is at least about 95%, 96%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO: 32; (v) a VHcomprising an amino acid sequence that is at least about 95%, 96%, 97%,98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 39,and a VL comprising an amino acid sequence that is at least about 95%,96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQID NO: 40; (vi) a VH comprising an amino acid sequence that is at leastabout 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acidsequence of SEQ ID NO: 47, and a VL comprising an amino acid sequencethat is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to theamino acid sequence of SEQ ID NO: 48; (vii) a VH comprising an aminoacid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO: 55, and a VLcomprising an amino acid sequence that is at least about 95%, 96%, 97%,98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 56;(viii) a VH comprising an amino acid sequence that is at least about95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence ofSEQ ID NO: 63, and a VL comprising an amino acid sequence that is atleast about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acidsequence of SEQ ID NO: 64; or (ix) a VH comprising an amino acidsequence that is at least about 95%, 96%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO: 71, and a VLcomprising an amino acid sequence that is at least about 95%, 96%, 97%,98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 72.5. The antibody of claim 3, wherein the antibody is an IgG, particularlyan IgG₁, antibody.
 6. The antibody of claim 3, wherein the antibody is afull-length antibody.
 7. The antibody of claim 3, wherein the antibodyis an antibody fragment selected from the group of an Fv molecule, ascFv molecule, a Fab molecule, and a F(ab′)₂ molecule.
 8. The antibodyof claim 3, wherein the antibody is a multispecific antibody.
 9. Abispecific antigen binding molecule, comprising (a) a first antigenbinding moiety that binds to a first antigen, wherein the first antigenis HLA-A2/WT1 and the first antigen binding moiety comprises (i) a heavychain variable region (VH) comprising a heavy chain complementarydetermining region (HCDR) 1 of SEQ ID NO: 1, a HCDR 2 of SEQ ID NO: 2,and a HCDR 3 of SEQ ID NO: 3, and a light chain variable region (VL)comprising a light chain complementarity determining region (LCDR) 1 ofSEQ ID NO: 4, a LCDR 2 of SEQ ID NO: 5 and a LCDR 3 of SEQ ID NO: 6;(ii) a VH comprising a HCDR 1 of SEQ ID NO: 9, a HCDR 2 of SEQ ID NO:10, and a HCDR 3 of SEQ ID NO: 11, and a VL comprising a LCDR 1 of SEQID NO: 12, a LCDR 2 of SEQ ID NO: 13 and a LCDR 3 of SEQ ID NO: 14;(iii) a VH comprising a HCDR 1 of SEQ ID NO: 17, a HCDR 2 of SEQ ID NO:18, and a HCDR 3 of SEQ ID NO: 19, and a VL comprising a LCDR 1 of SEQID NO: 20, a LCDR 2 of SEQ ID NO: 21 and a LCDR 3 of SEQ ID NO: 22; (iv)a VH comprising a HCDR 1 of SEQ ID NO: 25, a HCDR 2 of SEQ ID NO: 26,and a HCDR 3 of SEQ ID NO: 27, and a VL comprising a LCDR 1 of SEQ IDNO: 28, a LCDR 2 of SEQ ID NO: 29 and a LCDR 3 of SEQ ID NO: 30; (v) aVH comprising a HCDR 1 of SEQ ID NO: 33, a HCDR 2 of SEQ ID NO: 34, anda HCDR 3 of SEQ ID NO: 35, and a VL comprising a LCDR 1 of SEQ ID NO:36, a LCDR 2 of SEQ ID NO: 37 and a LCDR 3 of SEQ ID NO: 38; (vi) a VHcomprising a HCDR 1 of SEQ ID NO: 41, a HCDR 2 of SEQ ID NO: 42, and aHCDR 3 of SEQ ID NO: 43, and a VL comprising a LCDR 1 of SEQ ID NO: 44,a LCDR 2 of SEQ ID NO: 45 and a LCDR 3 of SEQ ID NO: 46; (vii) a VHcomprising a HCDR 1 of SEQ ID NO: 49, a HCDR 2 of SEQ ID NO: 50, and aHCDR 3 of SEQ ID NO: 51, and a VL comprising a LCDR 1 of SEQ ID NO: 52,a LCDR 2 of SEQ ID NO: 53 and a LCDR 3 of SEQ ID NO: 54; (viii) a VHcomprising a HCDR 1 of SEQ ID NO: 57, a HCDR 2 of SEQ ID NO: 58, and aHCDR 3 of SEQ ID NO: 59, and a VL comprising a LCDR 1 of SEQ ID NO: 60,a LCDR 2 of SEQ ID NO: 61 and a LCDR 3 of SEQ ID NO: 62; or (ix) a VHcomprising a HCDR 1 of SEQ ID NO: 65, a HCDR 2 of SEQ ID NO: 66, and aHCDR 3 of SEQ ID NO: 67, and a VL comprising a LCDR 1 of SEQ ID NO: 68,a LCDR 2 of SEQ ID NO: 69 and a LCDR 3 of SEQ ID NO: 70, and (b) asecond antigen binding moiety which specifically binds to a secondantigen.
 10. The bispecific antigen binding molecule of claim 9, whereinthe first antigen binding moiety comprises (i) a VH comprising an aminoacid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO: 7, and a VLcomprising an amino acid sequence that is at least about 95%, 96%, 97%,98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 8;(ii) a VH comprising an amino acid sequence that is at least about 95%,96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQID NO: 15, and a VL comprising an amino acid sequence that is at leastabout 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acidsequence of SEQ ID NO: 16; (iii) a VH comprising an amino acid sequencethat is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to theamino acid sequence of SEQ ID NO: 23, and a VL comprising an amino acidsequence that is at least about 95%, 96%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO: 24; (iv) a VHcomprising an amino acid sequence that is at least about 95%, 96%, 97%,98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 31,and a VL comprising an amino acid sequence that is at least about 95%,96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQID NO: 32; (v) a VH comprising an amino acid sequence that is at leastabout 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acidsequence of SEQ ID NO: 39, and a VL comprising an amino acid sequencethat is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to theamino acid sequence of SEQ ID NO: 40; (vi) a VH comprising an amino acidsequence that is at least about 95%, 96%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO: 47, and a VLcomprising an amino acid sequence that is at least about 95%, 96%, 97%,98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 48;(vii) a VH comprising an amino acid sequence that is at least about 95%,96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQID NO: 55, and a VL comprising an amino acid sequence that is at leastabout 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acidsequence of SEQ ID NO: 56; (viii) a VH comprising an amino acid sequencethat is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to theamino acid sequence of SEQ ID NO: 63, and a VL comprising an amino acidsequence that is at least about 95%, 96%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO: 64; or (ix) a VHcomprising an amino acid sequence that is at least about 95%, 96%, 97%,98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 71,and a VL comprising an amino acid sequence that is at least about 95%,96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQID NO:
 72. 11. The bispecific antigen binding molecule of claim 9 or 10,wherein the second antigen is CD3, particularly CD3ε.
 12. The bispecificantigen binding molecule of claim 11, wherein the second antigen bindingmoiety comprises (i) a VH comprising a HCDR 1 of SEQ ID NO: 115, a HCDR2 of SEQ ID NO: 116, and a HCDR 3 of SEQ ID NO: 117, and a VL comprisinga LCDR 1 of SEQ ID NO: 118, a LCDR 2 of SEQ ID NO: 119 and a LCDR 3 ofSEQ ID NO: 120; or (ii) a VH comprising a HCDR 1 of SEQ ID NO: 130, aHCDR 2 of SEQ ID NO: 131, and a HCDR 3 of SEQ ID NO: 132, and a VLcomprising a LCDR 1 of SEQ ID NO: 133, a LCDR 2 of SEQ ID NO: 134 and aLCDR 3 of SEQ ID NO:
 135. 13. The bispecific antigen binding molecule ofclaim 12, wherein the second antigen binding moiety comprises (i) a VHcomprising an amino acid sequence that is at least about 95%, 96%, 97%,98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 21,and a VL comprising an amino acid sequence that is at least about 95%,96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQID NO: 22; or (ii) a VH comprising an amino acid sequence that is atleast about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acidsequence of SEQ ID NO: 136, and a VL comprising an amino acid sequencethat is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to theamino acid sequence of SEQ ID NO:
 137. 14. The bispecific antigenbinding molecule of claim 9, wherein the first and/or the second antigenbinding moiety is a Fab molecule.
 15. The bispecific antigen bindingmolecule of claim 9, wherein the second antigen binding moiety is a Fabmolecule wherein the variable domains VL and VH or the constant domainsCL and CH1, particularly the variable domains VL and VH, of the Fablight chain and the Fab heavy chain are replaced by each other.
 16. Thebispecific antigen binding molecule of claim 9, wherein the firstantigen binding moiety is a Fab molecule wherein in the constant domainthe amino acid at position 124 is substituted independently by lysine(K), arginine (R) or histidine (H) (numbering according to Kabat) andthe amino acid at position 123 is substituted independently by lysine(K), arginine (R) or histidine (H) (numbering according to Kabat), andin the constant domain CH1 the amino acid at position 147 is substitutedindependently by glutamic acid (E), or aspartic acid (D) (numberingaccording to Kabat EU index) and the amino acid at position 213 issubstituted independently by glutamic acid (E), or aspartic acid (D)(numbering according to Kabat EU index).
 17. The bispecific antigenbinding molecule of claim 9, wherein the first and the second antigenbinding moiety are fused to each other, optionally via a peptide linker.18. The bispecific antigen binding molecule of claim 9, wherein thefirst and the second antigen binding moiety are each a Fab molecule andwherein either (i) the second antigen binding moiety is fused at theC-terminus of the Fab heavy chain to the N-terminus of the Fab heavychain of the first antigen binding moiety, or (ii) the first antigenbinding moiety is fused at the C-terminus of the Fab heavy chain to theN-terminus of the Fab heavy chain of the second antigen binding moiety.19. The bispecific antigen binding molecule of claim 9, comprising athird antigen binding moiety.
 20. The bispecific antigen bindingmolecule of claim 19, wherein the third antigen moiety is identical tothe first antigen binding moiety.
 21. The bispecific antigen bindingmolecule of claim 9, comprising an Fc domain composed of a first and asecond subunit.
 22. The bispecific antigen binding molecule of claim 21,wherein the first, the second and, where present, the third antigenbinding moiety are each a Fab molecule; and wherein either (i) thesecond antigen binding moiety is fused at the C-terminus of the Fabheavy chain to the N-terminus of the Fab heavy chain of the firstantigen binding moiety and the first antigen binding moiety is fused atthe C-terminus of the Fab heavy chain to the N-terminus of the firstsubunit of the Fc domain, or (ii) the first antigen binding moiety isfused at the C-terminus of the Fab heavy chain to the N-terminus of theFab heavy chain of the second antigen binding moiety and the secondantigen binding moiety is fused at the C-terminus of the Fab heavy chainto the N-terminus of the first subunit of the Fc domain; and wherein thethird antigen binding moiety, where present, is fused at the C-terminusof the Fab heavy chain to the N-terminus of the second subunit of the Fcdomain.
 23. The bispecific antigen binding molecule of claim 21, whereinthe Fc domain is an IgG, particularly an IgG₁, Fc domain.
 24. Thebispecific antigen binding molecule of any one of claim 21 claim 21,wherein the Fc domain is a human Fc domain.
 25. The bispecific antigenbinding molecule of claim 21, wherein an amino acid residue in the CH3domain of the first subunit of the Fc domain is replaced with an aminoacid residue having a larger side chain volume, thereby generating aprotuberance within the CH3 domain of the first subunit which ispositionable in a cavity within the CH3 domain of the second subunit,and an amino acid residue in the CH3 domain of the second subunit of theFc domain is replaced with an amino acid residue having a smaller sidechain volume, thereby generating a cavity within the CH3 domain of thesecond subunit within which the protuberance within the CH3 domain ofthe first subunit is positionable.
 26. The bispecific antigen bindingmolecule of claim 21, wherein the Fc domain comprises one or more aminoacid substitution that reduces binding to an Fc receptor and/or effectorfunction.
 27. One or more isolated polynucleotide encoding the antibodyof claim
 3. 28. One or more vector, particularly expression vector,comprising the polynucleotide(s) of claim
 27. 29. A host cell comprisingthe polynucleotide(s) of claim
 27. 30. A method of producing an antibodythat binds to HLA-A2/WT1, comprising the steps of a) culturing the hostcell of claim 29 under conditions suitable for the expression of theantibody and b) optionally recovering the antibody.
 31. An antibody thatbinds to HLA-A2/WT1, produced by the method of claim
 30. 32. Apharmaceutical composition comprising the antibody of claim 3 and apharmaceutically acceptable carrier.
 33. A method of treating a diseasein an individual, comprising administering to said individual atherapeutically effective amount of a composition comprising theantibody of claim 3 in a pharmaceutically acceptable form.
 34. One ormore isolated polynucleotide encoding the bispecific antigen bindingmolecule of claim
 9. 35. One or more vector, particularly expressionvector, comprising the polynucleotide(s) of claim
 34. 36. A host cellcomprising the polynucleotide(s) of claim
 34. 37. A method of producinga bispecific antigen binding molecule that binds to HLA-A2/WT1 and asecond antigen, comprising the steps of a) culturing the host cell ofclaim 36 under conditions suitable for the expression of the bispecificantigen binding molecule and b) optionally recovering the bispecificantigen binding molecule.
 38. An bispecific antigen binding moleculethat binds to HLA-A2/WT1 and a second antigen, produced by the method ofclaim
 37. 39. A pharmaceutical composition comprising the bispecificantigen binding molecule of claim 9 and a pharmaceutically acceptablecarrier.
 40. A method of treating a disease in an individual, comprisingadministering to said individual a therapeutically effective amount of acomposition comprising the bispecific antigen binding molecule of claim9 in a pharmaceutically acceptable form.
 41. The antibody of claim 1,wherein the antibody is an IgG, particularly an IgG₁, antibody.
 42. Theantibody of claim 1, wherein the antibody is a full-length antibody. 43.The antibody of claim 1, wherein the antibody is an antibody fragmentselected from the group of an Fv molecule, a scFv molecule, a Fabmolecule, and a F(ab′)₂ molecule.
 44. The antibody of claim 1, whereinthe antibody is a multispecific antibody.